Transformation of C3H/10T1/2 mouse embryo cells to focus formation and anchorage independence by insoluble lead chromate but not soluble calcium chromate: relationship to mutagenesis and internalization of lead chromate particles.

The genotoxicity of soluble and insoluble hexavalent chromium compounds was studied in mammalian cell assays which detect base substitution, deletion, addition, and frameshift mutations [6-thioguanine resistance in Chinese hamster ovary cells], primarily base substitution mutations [ouabain resistance in Chinese hamster ovary and C3H/10T1/2 Cl 8 mouse embryo fibroblasts (10T1/2)] and morphological transformation [focus formation] in 10T1/2 cells. Soluble hexavalent CaCrO4, administered in either acute (5-h) or subacute (24-h) dosing regimens, induced dose-dependent cytotoxicity and mutation to 6-thioguanine resistance in Chinese hamster ovary cells but no mutation to ouabain resistance or focus formation in transformation assays, although the acute treatment induced a high frequency of conversion of 10T1/2 cells to adipocytes. Cell lines established from cloned adipocytic cells were not morphologically transformed and did not grow in soft agarose. PbCrO4 did not induce mutation to either 6-thioguanine or ouabain resistance but did induce a reproducible dose-dependent, low frequency of focus formation in 10T1/2 cells. Cell lines established from PbCrO4-induced foci stably formed foci when coseeded with 10T1/2 cells, had 3-5-fold increased saturation densities relative to nontransformed 10T1/2 cells, and formed colonies in soft agarose, indicating their likelihood to be neoplastic. Long term exposure of 10T1/2 cells to either CaCrO4 or PbCl2, even at 85% cytotoxic concentrations, or pretreatment of cells with either CaCrO4 or PbCl2 followed by treatment with the alternate compound, did not induce morphological transformation. Treatment of cells with insoluble hexavalent PbCrO4 resulted in progressive and extensive vacuolization of cells in contact with the particles. Progressive cytoplasmic engulfment of PbCrO4 particles was observed using scanning electron microscopy, although PbCrO4 particles were not observed inside vacuoles. These results indicate that the soluble clastogens K2Cr2O7 and CaCrO4 were probably mutagenic by a non-base substitution mechanism but could not transform 10T1/2 cells. In contrast, PbCrO4 was not detectably mutagenic but induced transformation, which could not be explained solely by acute or chronic exposure to dissolution products of either lead or chromate alone. Since PbCrO4 particles were found to be intracytoplasmic in extensively vacuolated cells, we suggest that the unique physiochemical properties of PbCrO4 particles, leading to their internalization and the resultant associated cellular stress response, may be related to the transformation induced by this compound.

Preferential DNA-protein cross-linking by NiCl2 in magnesium-insoluble regions of fractionated Chinese hamster ovary cell chromatin.

Intracellular nickel ions (Ni2+) have been shown to cause single-strand breaks in DNA, that were rapidly repaired, and DNA-protein cross-links, that persisted for at least 24 h following removal of extracellular ionic nickel. In this study, we have used the techniques of alkaline elution, chromatin fractionation, and sodium dodecyl sulfate:polyacrylamide gel electrophoresis to examine the DNA-protein cross-linking induced by NiCl2 in Chinese hamster ovary cells. Continuous treatment of logarithmically growing Chinese hamster ovary cells with 2.5 mM NiCl2 in complete medium resulted in DNA single-strand breaks within 1 h, followed by a time-dependent increase in the induction of DNA-protein cross-links at 2, 3, and 6 h. Since the entry of nickel into cells was maximal within 2 h of exposure, the time delay for the formation of DNA-protein cross-links was not limited by metal uptake. The nickel-induced DNA-protein cross-linking appeared to require active cell cycling, since single-strand breaks but no cross-linking could be detected in confluent cells treated with 1, 2.5, or 5 mM NiCl2 for 3 h. DNA-protein cross-linking induced by nickel occurred in late S phase of the cell cycle. High-molecular-weight nonhistone chromatin proteins and possibly histone H1 migrating at the Mr 30,000 range became cross-linked to DNA after treatment of cells with NiCl2. All nickel-cross-linked proteins were concentrated in the magnesium-insoluble regions of fractionated chromatin and were stable to urea, 2-mercaptoethanol, and Nonidet P-40. Some proteins (Mr 48,000, 52,000, 55,000, 70,000, and 95,000), the association of which with DNA was also stable to Sarkosyl, salt, and EDTA, were detectable in DNA rigorously fractionated from untreated cells. Nickel therefore appeared to cause the cross-linking of proteins that normally reside in close association with DNA. Alterations of the normal association of these proteins with DNA by nickel may be an early event in the nickel transformation process.

Study of the ability of phenacetin, acetaminophen, and aspirin to induce cytotoxicity, mutation, and morphological transformation in C3H/10T1/2 clone 8 mouse embryo cells.

Use of the analgesic compounds acetylsalicylic acid (aspirin), phenacetin, and acetaminophen has been correlated with increased risk of renal cancer in humans. Hence, we studied these compounds for ability to induce cytotoxicity, mutation to ouabain resistance, and morphological transformation in cultured C3H/10T1/2 clone 8 (10T1/2) mouse embryo cells. All three compounds were cytotoxic from 0.5-mg/ml to 2-mg/ml concentrations as evidenced by decreased plating efficiency. None of the compounds induced detectable base substitution mutations to ouabain resistance even at cytotoxic concentrations. Aspirin did not induce morphological transformation. Both phenacetin and acetaminophen induced low but concentration-dependent numbers of atypical, weak type II morphologically transformed foci; at equimolar concentrations, phenacetin was 1.1- to 3.0-fold more active in inducing these foci. Neither phenacetin nor acetaminophen was cotransforming with 3-methylcholanthrene, and neither compound promoted cell transformation when added to 3-methylcholanthrene-initiated 10T1/2 cells. The focus-inducing potency of both compounds was increased by addition of an Arochlor-induced hamster liver S9 fraction as an exogenous metabolizing system. However, seven putative metabolites of phenacetin and acetaminophen that were tested--N-hydroxyphenacetin, p-phenetidine, p-aminophenol, p-nitrosophenol, benzoquinone, acetamide, and N-acetyl-p-benzoquinoneimine--were inactive in transformation assays at the concentrations reducing plating efficiency of treated cells to 50% of the plating efficiency of nontreated (control) cells. Several acetaminophen- and phenacetin-induced foci were cloned, expanded into cell lines, and characterized. These cell lines stably formed type II foci when maintained at confluence for 2 to 4 wk in reconstruction experiments with nontransformed 10T1/2 cells; however, they did not exhibit significantly increased saturation density compared to 10T1/2 cells, and they did not grow in soft agarose. These results suggest that metabolic intermediates of high concentrations of phenacetin and acetaminophen induce a low frequency of nonneoplastic morphological transformation of 10T1/2 mouse embryo cells.

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.

Enhanced expression of c-myc and decreased expression of c-fos protooncogenes in chemically and radiation-transformed C3H/10T1/2 Cl 8 mouse embryo cell lines.

c-abl, c-fos, c-Ha-ras, c-myc, and c-mos were expressed whereas c-sis, c-fms, c-rel, c-src, and c-myb expression was not detectable in C3H/10T1/2 Cl 8 (10T1/2) cells and in eight chemically and radiation-transformed 10T1/2 cell lines. The expression of c-abl, c-fos, c-Ha-ras, and c-myc was growth-related in nontransformed 10T1/2 cells. c-abl and c-fos expression increased at confluence by 5- and 9-fold, respectively, compared to that in log phase cells. c-Ha-ras and c-myc transcripts were most abundant in log phase cells and decreased by 70 and 50%, respectively, in confluent cells. There were no significant growth-related changes in the expression of c-Ha-ras, c-myc, or c-abl in methylcholanthrene-transformed Cl 15 cells. The c-fos transcript was not detected in Cl 15 cell cultures. c-abl, c-fos, c-ras, and c-myc were expressed in whole C3H mouse embryo tissue, mouse liver, and 10T1/2 cells. Sizes of these protooncogene transcripts in 10T1/2 cells were the same as those in whole embryo tissue, except that 10T1/2 cells did not express the 8.2-kilobase abl transcript. At subconfluence, equivalent low levels of c-mos expression were observed in nontransformed and in the eight transformed 10T1/2 cell lines. The level of c-abl expression was similar in the nontransformed and in the eight transformed cell lines, but there was a new 8.2-kilobase transcript in the transformed MCA Cl 15 cell line. c-fos was expressed in 10T1/2 cells but was not detectable or greatly reduced in eight transformed cell lines. c-Ha-ras was expressed to a similar extent in eight transformed cell lines and in nontransformed 10T1/2 cells. In the UVC-4 transformed cell line, extra 3.3-kilobase Ha-ras and 7.5-kilobase Ki-ras transcripts were observed. c-myc was expressed at 4- to 7-fold higher levels in six transformed cell lines compared to 10T1/2 cells. There were no major rearrangements in or amplification of the c-myc gene in three transformed cells overexpressing this gene 5-fold. These studies show that enhanced expression of c-myc and decreased expression of c-fos correlate with the chemically and radiation transformed states of 10T1/2 cells. Changes in c-fos and c-myc oncogene expression may be casually linked to late stages of neoplastic transformation in these chemically and radiation transformed 10T1/2 cell lines.

Increased expression of the glucose-regulated gene encoding the Mr 78,000 glucose-regulated protein in chemically and radiation-transformed C3H 10T1/2 mouse embryo cells.

Expression of the gene coding for the Mr 78,000 glucose-regulated protein (GRP78) was examined in nontransformed and chemically and radiation-transformed C3H 10T1/2 Cl 8 mouse embryo cells. When cells were grown in complete medium with 10% fetal bovine serum, GRP78 mRNA was increased 4- to 9-fold in 3-methylcholanthrene (MCA; Clones 15 and 16)-, bleomycin (Bleo 1)-, and ultraviolet light (UV-C3)-transformed cell lines compared to nontransformed 10T1/2 clone 8 cells (Cl 8) at similar cell number and growth phase. Increased steady-state levels of GRP78 protein in MCA Cl 15 compared to Cl 8 cells were confirmed by 2-dimensional gel electrophoresis. Under these conditions transformed MCA Cl 15 exhibited increased GRP78 RNA within 24 h after addition of fresh glucose-containing medium, whereas nontransformed Cl 8 cells did not increase expression of this gene even after 5 days of culture in conditioned medium. Incubation of Cl 8 and MCA Cl 15 in glucose-free medium for 16 h caused a 3- and 15-fold induction of GRP78 RNA, respectively. In addition, chemically transformed cells were highly sensitive to glucose deprivation and responded by rounding up and detaching from the substratum. Cl 8 cells exhibited no such sensitivity to glucose deprivation. These results extend earlier reports on virally transformed cells to include chemically and radiation-transformed cells and expand earlier reports to include mRNA expression and 2-dimensional gel electrophoresis of GRP78 protein.

Inhibitors of prostaglandin synthesis inhibit human prostate tumor cell invasiveness and reduce the release of matrix metalloproteinases.

Eicosanoids modulate the interaction of tumor cells with various host components in cancer metastasis. Their synthesis involves the release of arachidonic acid (AA) from cellular phospholipids by phospholipase A2 (PLA2), followed by metabolism by cyclooxygenases (COXs) and lipooxygenases (LOXs). This study aimed to identify the pathway(s) of AA metabolism that are required for the invasion of prostate tumor cells. DU-145 and PC-3 human prostate cancer cell lines were used to test the effect of inhibitors of PLA2, COX, or LOX on the invasion of prostate tumor cells through Matrigel in vitro using the Boyden chamber assay and fibroblast-conditioned medium as the chemoattractant. We used nontoxic doses that did not inhibit simple cell motility and did not decrease clonogenic survival. All of the inhibitors caused a significant reduction in AA release from treated cells compared with control cells, which indicated that the treatments were biochemically active. Invasion through Matrigel was inhibited by the PLA2 inhibitor 4-bromophenacyl bromide (4-BPB), the general COX inhibitor ibuprofen (IB), and the highly selective COX-2 inhibitor NS398. Inhibition of cell invasiveness by 4-BPB (1.0 microM), IB (10.0 microM), and NS398 (10.0 microM) was reversed by the addition of prostaglandin E2 (PGE2). PGE2 alone, however, did not stimulate invasiveness, which suggests that its production is necessary for rendering the cells invasive-permissive but not sufficient for inducing invasiveness. In contrast, we found no significant inhibition of invasion of prostate tumor cells treated with esculetin (1.0 microM) or nordihydroguiaretic acid (1.0 microM), which are specific inhibitors of LOX. We also tested the effect of 4-BPB, IB, NS398, and esculetin on the secretion of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), as key enzymes in the proteolysis of Matrigel during invasion, using gelatin zymograms and Western blots. Cells that received 4-BPB, IB, or NS398, but not esculetin showed a significant reduction in the levels of proMMP-2, MMP-9, and proMMP-9 in the culture medium. DU-145 cells did not secrete TIMP-1, and the drugs did not alter the secretion of TIMP-2. This work highlights the role played by COX in disturbing the balance between MMPs and TIMPs in prostate cancer cells, and it points to the potential use of COX inibitors, especially COX-2 selective inhibitors, in the prevention and therapy of prostate cancer invasion.

Prevention of brefeldin A-induced resistance to teniposide by the proteasome inhibitor MG-132: involvement of NF-kappaB activation in drug resistance.

Brefeldin A, an agent that disrupts protein transport from the endoplasmic reticulum to the Golgi, induces the expression of GRP78 and the activation of nuclear factor (NF)-kappaB in cells. Treatment of cells with brefeldin A causes the development of resistance to topoisomerase II-directed agents, such as etoposide and doxorubicin. In this study, we show that treatment of EMT6 mouse mammary tumor cells with brefeldin A strongly induces GRP78 mRNA (8.5-fold) and resistance to teniposide (VM26). Treatment with okadaic acid causes a minor increase in GRP78 mRNA (2.1-fold) yet still induces resistance to VM26 as effectively as brefeldin A. In contrast, cells treated with castanospermine show a moderate increase in GRP78 mRNA (3.9-fold) but no resistance to VM26. These data imply that GRP78 induction does not mediate the development of drug resistance. An alternative mechanism of drug resistance may involve activation of the transcription factor, NF-kappaB, and we show that both brefeldin A and okadaic acid activate NF-kappaB in EMT6 cells. Furthermore, we demonstrate that treatment with the proteasome inhibitor MG-132 blocks the activation of NF-kappaB and prevents the development of resistance to VM26 induced by brefeldin A. Collectively, these results suggest that the resistance to VM26 in EMT6 cells treated with brefeldin A is mediated by the activation of NF-kappaB rather than the induction of GRP78. Our results also suggest that inhibition of NF-kappaB activation in tumor cells may increase the efficacy of topoisomerase II-directed agents in chemotherapy.

Loss of uteroglobin expression in prostate cancer: relationship to advancing grade.

We have shown previously that the secretory protein uteroglobin (UG) is highly expressed in normal human prostate tissue but this expression is either lost or altered in human prostate cancer cell lines. Treatment of these cell lines with recombinant human UG inhibits their ability to invade human reconstituted basement membrane by up to 90%, implying that the loss of normal UG expression may be related to the invasive potential of prostate cancer. Because invasion represents a critical step in metastasis, the expression patterns of UG could provide a unique and relevant indicator of cancer progression. In this study, we present the immunohistochemical analyses of fresh frozen prostate tissues from surgical specimens taken from 50 patients. Eight slides per patient were analyzed for UG staining. Slides from 26 patients showed evidence of prostate cancer, whereas slides from the remaining 24 patients showed only benign glands. The results demonstrate UG immunoreactivity in normal prostate, benign prostatic hyperplasia, and prostatic atrophy; low but clearly positive expression in prostatic intraepithelial neoplasia; positive expression in cancerous glands of Gleason's pattern

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.

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)

Differential sensitivity of chromium-mediated DNA interstrand crosslinks and DNA-protein crosslinks to disruption by alkali and EDTA.

Some compounds of hexavalent chromium are well-established carcinogens. Chromium enters mammalian cells in the hexavalent form and is reduced to chromium (III). Treatment of purified DNA with chromium (III) produces DNA-DNA interstrand crosslinks (DDC) which obstruct the progression of DNA polymerases in vitro. DDC were also detected in chromate-treated cultured normal human lung cells using the renaturing agarose gel electrophoresis (RAGE) assay and correlated with base-specific inhibition of DNA replication. Curiously, DDC have gone undetected in studies of cultured cells using the alkaline elution (AE) technique, whereas chromium-mediated DNA-protein crosslinks (DPC) were readily detected by AE. We tested the hypothesis that AE conditions [60 mM tetraethyl ammonium hydroxide (TEA), 20 mM EDTA, pH 12.6, for 16 h at room temperature] dissociate DDC but not DPC using chromium(III)-treated plasmid DNA and the RAGE assay. Dose-dependent chromium-induced DDC were unaffected by TEA (pH 11.8) alone or by more rigorous alkaline denaturation conditions (200 mM NaOH, pH 13.5, for 16 h). DDC were, however, completely disrupted by EDTA (pH 12.6) alone or the combination of TEA and EDTA (pH 12.6). In contrast, DPC remained largely intact under these conditions. Therefore, past AE-based studies which have failed to detect chromium-induced DDC do not prove the absence of this lesion. AE may not be suitable for detecting DDC induced by EDTA-chelatable agents such as metals.

Sensitive quantitation of chromium-DNA adducts by inductively coupled plasma mass spectrometry with a direct injection high-efficiency nebulizer.

A novel method is described for the sensitive detection of chromium-DNA adducts. Chromium-DNA adducts were determined in 1 microgram of DNA from normal human lung fibroblasts exposed to sodium chromate using microscale flow injection analysis with a direct injection high-efficiency nebulizer and inductively coupled plasma mass spectrometry detection. The frequency of Cr-DNA adducts increased in a dose-dependent sigmoidal manner, indicating saturation and toxicity. The low detection limits (on the order of parts per trillion) allows the detection of as few as two Cr adducts per 10,000 bases, which, coupled with the small DNA sample requirement, makes this technique suitable for measuring metal-DNA adducts as biomarkers of exposure to toxic and carcinogenic metals such as Cr, in cultured cells, animals, and humans.

Apoptosis and P53 induction in human lung fibroblasts exposed to chromium (VI): effect of ascorbate and tocopherol.

Some forms of hexavalent chromium [Cr(VI)] are known to cause damage to respiratory tract tissue, and are thought to be human lung carcinogens. Because Cr(VI) is mutagenic and carcinogenic at doses that evoke cell toxicity, the objective of these experiments was to examine the effect of Cr(VI) on the growth, survival, and mode of cell death in normal human lung fibroblasts (HLF cells). DNA adduct formation was monitored as a marker for bioavailability of genotoxic chromium. We also examined the modulation of these endpoints by vitamins C and E. Long-term Cr(VI) exposures were employed, which decreased clonogenic cell survival by 25% to 95% in a dose-dependent manner. The predominant cellular response to Cr(VI) was growth arrest. We found that Cr(VI) caused up to 20% of HLF cells to undergo apoptosis, and documented apoptotic morphology and the phagocytosis of apoptotic bodies by neighboring cells. P53 levels increased 4- to 6-fold in chromium-treated cells. In contrast with previous studies using CHO cells, the present study using HLFs found that pretreatment with either vitamin C or E did not exhibit a significant effect on Cr-induced apoptosis or clonogenic survival. In addition, pretreatment with vitamin C did not affect the p53 induction observed after chromium treatment. Neither vitamin had any effect on Cr-DNA adduct formation. These data indicate that although pretreatment with vitamin C or E alters the spectrum of cellular and/or genetic lesions induced by chromium(VI), neither vitamin altered the initiation or progression of apoptosis in diploid human lung cells.

Morphological and neoplastic transformation of C3H/10T1/2 Cl 8 mouse embryo cells by insoluble carcinogenic nickel compounds.

We studied induction of cytotoxicity and morphological transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblasts by soluble and insoluble carcinogenic nickel compounds. Soluble nickel sulfate and nickel chloride caused dose-dependent cytotoxicity in the concentration range from 0.5 microM to 100 microM after 48 hr treatments, but neither compound induced morphological transformation even at concentrations causing up to 94% cytotoxicity. Insoluble nickel subsulfide, nickel monosulfide, and nickel oxide caused dose-dependent cytotoxicity and a low, dose-dependent frequency of morphological transformation in the concentration ranges from 0.5 to 40 microM, 5 to 50 microM, and 50 to 400 microM, respectively, after 48 hr exposure of cells to these compounds. Foci were predominantly of type II morphology; type III foci were rare. The insoluble nickel compounds studied caused no induction of base substitution mutations to ouabain resistance in 10T1/2 cells over concentration ranges that induced morphological transformation. Nickel subsulfide and nickel monosulfide were taken into cells by phagocytosis, since particles were visible in intracytoplasmic vacuoles. Numerous nickel oxide particles were found associated with cells, but true phagocytic uptake was difficult to detect since no vacuoles were observed. We twice cloned type II and type III foci induced by insoluble nickel compounds, established independent cell lines, and characterized their phenotypes. Four of seven of these cell lines had three- to fourfold increased saturation densities compared to 10T1/2 cells, formed type II and type III foci in reconstruction assays, and grew in soft agarose. One cell line induced by nickel oxide formed tumors in nude mice. These data indicate that insoluble carcinogenic nickel compounds induced type II foci in 10T1/2 cells, some of which were tumorigenic, and that the 10T1/2 cell system is suitable for studying mechanisms of nickel compound-induced morphological transformation in mammalian cells.

Prostaglandin A1 inhibits stress-induced NF-kappaB activation and reverses resistance to topoisomerase II inhibitors.

Stress conditions associated with solid tumors lead to the formation of heterogeneous tumor cell subpopulations and insensitivity to cancer chemotherapeutics. In this report, we show that EMT6 mouse mammary tumor cells treated with the chemical stress, brefeldin A (BFA), or the physiological stress, hypoxia, develop resistance to the topoisomerase II (topoII) inhibitors teniposide and etoposide. BFA and hypoxia treatment did not alter intracellular drug concentrations, topoll protein levels, or inhibit topoII activity. BFA and hypoxia did cause the activation of the nuclear transcription factor NF-kappaB. We demonstrate that pretreatment with the synthetic cyclopentenone prostaglandin A1 (PGA1) inhibits stress-induced NF-kappaB activation and reverses BFA- and hypoxia-induced resistance. The reversal of BFA-induced resistance can occur when PGA1 is administered either before or several hours after the induction of stress. Taken together, these data support the involvement of NF-kappaB in stress-induced drug resistance, show that pharmacologic inhibitors of NF-kappaB can disrupt the biological consequences of stress, and imply that inhibitors of NF-kappaB may be useful agents to enhance the clinical efficacy of topoII-directed chemotherapeutics.

Chromium-induced genotoxicity and apoptosis: relationship to chromium carcinogenesis (review).

The adverse health effects linked with chromium (Cr) exposure, the role of solubility and chemical speciation of Cr compounds, and the diverse cellular and molecular effects of Cr make the study of Cr carcinogenesis and toxicology very interesting and complex. Certain Cr compounds are prominent metal carcinogens in both occupational and environmental settings. Inhaled particulate forms of hexavalent Cr [Cr(VI)] cause lung cancer as well as lung toxicity. Some of the important factors in determining the biological outcome of Cr exposure include the bioavailability, chemical speciation and solubility of Cr compounds, intracellular reduction, and interaction of Cr with DNA. The stable oxidation states of Cr found in nature are Cr(III) and Cr(VI). Cr(III) is unable to enter cells but Cr(VI) enters into cells through membrane anionic transporters. Intracellular Cr(VI) is metabolically reduced to the ultimate Cr(III). Cr(VI) does not react with macromolecules such as DNA, RNA, proteins and lipids. However, both Cr(III) and the reductional intermediate Cr(V) are capable of co-ordinate covalent interactions with macromolecules. At the genomic level, Cr genotoxicity manifests as gene mutations, several types of DNA lesions and inhibition of macromolecular synthesis. At the cellular level, Cr exposure may lead to cell cycle arrest, apoptosis, premature terminal growth arrest, or neoplastic transformation. Cr-induced DNA-DNA interstrand crosslinks (DDC), the tumor suppressor gene p53 and oxidative processes are some of the major factors that may play a significant role in determining the cellular outcome in response to Cr exposure. We have utilized cellular, molecular, pharmacological, and genetic approaches to understand the interrelationship between Cr-induced genotoxicity, apoptosis and carcinogenesis. This review is based on the results and inferences of this research. We hope this review will clarify existing concepts and also introduce novel perspectives in chromium carcinogenesis research.

Application of a modified 203Hg binding assay for metallothionein.

A sensitive and rapid method to estimate concentrations of functional metallothionein in small biological samples, based upon the acid stability of 203Hg binding and solubility of this protein in trichloroacetic acid is described. Sephadex G-10 minicolumns supported in centrifuge tubes afforded separation and quantitation of isotope bound metallothionein from unbound metal. Elution of metallothionein bound 203Hg was achieved by short term-low speed centrifugation that segregated chelator-ligand complex into the eluate while unbound ligand remained in the gel. A well characterized standard of pure metallothionein protein was utilized to verify the specificity and sensitivity of the modified assay. Metallothionein levels were estimated by 203Hg binding in extracts of wild type and cadmium resistant Chinese hamster ovary cells treated with maximum tolerable concentrations of CdCl2. Similar separation methods demonstrated [35S]-cysteine incorporation into induced metallothionein. Additionally, induction of metallothionein was observed after treatment with particulate CdS but not crystalline NiS particles. These results demonstrate that the modified assay system is easily applied to serial measurement of metallothionein levels in multiple small biological samples.

DNA-protein cross-links induced by nickel compounds in intact cultured mammalian cells.

The carcinogenic activity of crystalline NiS has been attributed to phagocytosis and intracellular dissolution of the particles to yield Ni2+ which is thought to enter the nucleus and damage DNA. In this study the extent and type of DNA damage in Chinese hamster ovary CHO cells treated with various nickel compounds was assessed by alkaline elution. Both insoluble (crystalline NiS) and soluble (NiCl2) nickel compounds induced single strand breaks and DNA protein cross-links. The single strand breaks were repaired relatively quickly but the DNA-protein cross-links were present and still accumulating 24 h after exposure to nickel. Single strand breakage occurred at both non-cytotoxic and cytotoxic concentrations of nickel, however, DNA-protein cross-linking was absent when cells were exposed to toxic nickel levels. The concentration of nickel that induced DNA-protein cross-linking correlated with those metal concentrations that reversibly inhibited cellular replication.

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.