A method for assessing efficiency of bacterial cell disruption and DNA release.

BACKGROUND: DNA-based testing is becoming the preferred method both for identifying microorganisms and for characterizing microbial communities. However, no single DNA extraction method exists that is suitable for all types of microorganisms because bacteria are variable in their susceptibility to lysis by available extraction procedures. To develop and test new DNA extraction procedures, it would be helpful to determine their efficiencies. While the amount of extracted DNA can readily be measured by different methods, calculation of true efficiency requires knowledge of the initial amount of DNA in the starting bacterial sample, which cannot be done with precision by any existing method. In the process of developing a new extraction procedure, we developed a method that can be used to determine the total amount of both DNA and RNA in bacteria. The amount of DNA can be calculated from the amount of purines released after mild acid and alkali treatment. The amount of RNA in the same extract can also be calculated from the amount of ribonucleoside monophosphates. The released purines and ribonucleoside monophosphates can be quantified by absorbance using HPLC, with reference to appropriate standards. RESULTS: The acid/HPLC method was used to measure the efficiency of commonly used bead-beating and chemical protocols for releasing DNA from a particularly hardy organism, Mycobacterium smegmatis as well as several other species (Bacillus subtilis vegetative cells and spores; Francisella philomiragia; Pseudomonas aeruginosa; Moraxella catarrhalis; Bacillus thuringiensis; Staphylococcus aureus). Surprisingly large differences in efficiency between methods were found. CONCLUSIONS: The acid/HPLC method is a new tool to determine DNA extraction efficiencies and should aid in the development of improved protocols for releasing DNA from a broad range of microorganisms.

Expression of thymidylate synthase in human cells is an early G(1) event regulated by CDK4 and p16INK4A but not E2F.

Thymidylate synthase (TS) is the enzyme that catalyses the last step in de novo thymidylate synthesis. It is of interest clinically because it is an effective target for drugs such as 5-fluorouracil, often used in combination therapy. Despite a number of earlier reports indicating that TS is a cell cycle-dependent enzyme, this remains equivocal. Here, we show that in HCT116 cells synchronised by serum starvation, there is a clear dissociation between the expression of cyclin E (a well-characterised cell-cycle protein) and TS. Although both cyclin E and TS mRNA and protein increased during G(1), TS upregulation was delayed. Moreover, TS levels did not decrease following S-phase completion while cyclin E decreased sharply. Similarly, clear differences were seen between cyclin E and TS as asynchronously growing HCT116 cells were growth-inhibited by low-serum treatment. In contrast to previous reports using rodent cells, adenovirus-mediated over-expression of E2F1 and cyclin E in three human cell lines had no effect on TS. Cell-cycle progression was blocked by treatment of cells with pharmacological inhibitors of CDK2 and CDK4 and by ectopic expression of p16INK4A. Whereas CDK2 inhibition had no effect on TS levels, inhibition of CDK4 was associated with decreased TS protein levels. These results provide the first evidence that drugs targeting CDK4 may be useful with anti-TS drugs as combination therapy for cancer.

Constitutive expression of interleukin-8 by Mutatect cells markedly affects their tumor biology.

Interleukin-8 (IL-8) is a chemokine for neutrophils and an angiogenic factor. Human tumors that express IL-8 may exhibit intense neutrophil infiltration and increased vascularization. Mutatect cells are a murine fibrosarcoma that can be grown as subcutaneous tumors in syngeneic C57BL/6 mice. Since neutrophils are a source of cytotoxic and genotoxic species, we constructed Mutatect cell lines that constitutively express human IL-8 to explore the involvement of neutrophils in tumor biology and genetic instability. An IL-8/neo expression plasmid was stably transfected into Mutatect MC17-51 cells and clone MIL-4 was isolated. Tumors initiated with 5x10(5) MIL-4 cells grew very slowly compared with tumors from pure MC17-51 cells or from 0.5 to 4x10(5) MIL-4 cells mixed with 5x10(5) MC17-51 cells. Over 95% of cells recovered from slow-growing pure MIL-4 tumors lost the transgene as measured by loss of (i) resistance to G418, (ii) expression of IL-8 protein and (iii) IL-8-specific DNA sequences. When tumors from mixed cell types were examined, loss of the transgene did not occur; rather, IL-8 producing cells appeared to have some growth advantage. The neutrophil content of tumors (as measured by myeloperoxidase) was directly proportional to the level of IL-8 expressed at the time tumors were excised. As reported earlier, the frequency of mutations at the hypoxanthine phosphoribosyltransferase locus was also directly proportional to neutrophil content. To explain some of these biological findings, we postulate that early in development of pure MIL-4 tumors, genotoxic/cytotoxic neutrophils are attracted by IL-8, which in turn leads to loss of the transgene and to localized cytotoxicity of IL-8 producing cells. In mixed tumors, where the initial IL-8 concentration may be lower, tumors might become established more readily because fewer neutrophils may be attracted. This relatively simple experimental paradigm has revealed some of the complex biological changes that can occur as a result of IL-8 in tumors.

Nitric oxide donors induce large-scale deletion mutations in human lymphoblastoid cells: implications for mutations in T-lymphocytes from arthritis patients.

Rheumatoid arthritis (RA) is an inflammatory disease in which high levels of reactive nitrogen oxygen species (RNOS) may be present in the affected joints. RNOS are known to produce small-scale mutational events (transitions, transversions, small insertions, and small deletions) but the ability of these compounds to cause deletion of large segments of genomic DNA has not been previously determined. To address this question, a human lymphoblastoid cell line (WIL2-NS) was exposed to nitric oxide (NO)-donating drugs and hypoxanthine phosphoribosyltransferase (hprt)-negative clones were selected and analyzed by multiplex-PCR. Large-scale deletions accounted for 60-80% of hprt mutations arising in drug-treated cultures compared to 12% in untreated cultures (P-values of 0.006 and 0.0001, respectively, in two experiments). Deletion mutations in untreated cultures affected exon 9, whereas 75% of drug-induced deletion mutations affected exons 2, 3, and 9, and the remainder were very large, ranging from 26 to 1200 kbp. To compare this spectrum of NO-induced mutations in a lymphoblastoid line to that arising in vivo in arthritis patients, T-cells from RA patients, osteoarthritis (OA) patients, and controls were cloned and similarly analyzed. We previously showed that the overall frequency of Hprt mutant clones from patients is appreciably elevated compared to that of control subjects. Large-scale hprt deletions (0.5 to >26 kb) were detected in mutant T-cell clones from both RA and OA patients and also from control subjects. A total of 54 mutant clones from 16 RA patients and 19 mutant clones from 6 OA patients were studied. Of these, 6 clones (from 3 RA and 1 OA patient) had suffered large-scale deletions. A total of 9 control subjects were studied and 62 mutant clones were obtained. Of these, 19 had suffered large-scale deletions, arising in 7 of 9 control subjects. In conclusion, (1) RNOS are capable of inducing large-scale deletion mutations in a human lymphoblastoid cell line and (2) large-scale deletion mutations were found in 10-30% of T-cell clones from RA and OA patients and controls, which we hypothesize may be induced by RNOS.

Depletion of intracellular glutathione reduces mutations by nitric oxide-donating drugs.

The Mutatect system is a mouse tumor line in which mutations at the hypoxanthine phosphoribosyltransferase (Hprt) locus can be readily detected both in vitro and in vivo. We have previously shown that the nitric oxide-generating drugs, glyceryl trinitrate (GTN) and sodium nitroprusside (SNP), can induce mutations that are readily detected in these cells. In the present report, we have tested the effect of glutathione depletion by buthionine sulfoximine (BSO) on cytotoxicity and mutagenicity by these two drugs. Exposure for 24 h to either drug (123 microM GTN; 500 microM SNP) induced mutations with relatively little cytotoxicity. Pretreatment with 50 microM BSO for 24 h, and then removal at the time of GTN or SNP addition, enhanced cytotoxicity to a modest extent. However, mutagenicity induced by both GTN and SNP was largely abolished. BSO did not affect nitrite accumulation in the medium over a 24-h period, indicating no inhibition of bioactivation of GTN or SNP. Maintaining BSO in the medium for 24 h prior and throughout the period of exposure to GTN or SNP produced a similar effect on mutations. N-Acetylcysteine and oxothiazolidine-4-carboxylate, drugs that are used to increase intracellular glutathione, also blocked mutations. We postulate that a product of the reaction between nitric oxide and intracellular glutathione, such as GSNO or some species derived from it, is promutagenic.

Effect of dietary vitamin E on spontaneous or nitric oxide donor-induced mutations in a mouse tumor model.

BACKGROUND: Vitamin E, an antioxidant, has been investigated for its effect on cancer incidence in humans, but no firm conclusions about a protective effect can be drawn from these studies. Recently, we reported a statistically significant correlation in the Mutatect mouse tumor model between the number of neutrophils and the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus. We have now used this model to investigate vitamin E's effect on the hprt mutation rate. METHODS: Mutatect cells were grown in mice as subcutaneous tumors for 2-3 weeks, the tumor cells were recovered, and 6-thioguanine-resistant (i.e., hprt mutant) colonies were scored. Myeloperoxidase activity was used as a measure of neutrophil infiltration. Vitamin E (2 IU/kg body weight) was provided in the diet for 3-4 weeks. In some experiments, glyceryl trinitrate (100 mg/kg body weight) was also administered as a source of nitric oxide. All statistical tests were two-sided. RESULTS: Mouse tumors from the Mutatect MN-11 cell line exhibited a 3.2-fold higher median mutation frequency than the same cells in culture (P:<. 0001); vitamin E reduced this frequency by 24.9% (P: =.01). Mutatect TM-28-derived tumors (which secrete interleukin 8) were heavily infiltrated with neutrophils and had a correspondingly high mutation frequency; in two separate experiments, vitamin E reduced the median mutation frequency by 68.9% (P: =.0019) and 84.1% (P: =.011) and myeloperoxidase levels by 75.3% (P: =.0002) and 75.5% (P: =.026), respectively. Glyceryl trinitrate increased the mutation frequency in MN-11 tumors, and vitamin E reduced the median frequency by 61.4% (P: =.058). CONCLUSIONS: Dietary vitamin E afforded strong protection against both spontaneously arising and nitric oxide-induced mutations. Two separate protective mechanisms by vitamin E may be operating: scavenging of a nitric oxide-related genotoxic species and altering the infiltration of neutrophils into tumors.

Neutrophils, nitric oxide synthase, and mutations in the mutatect murine tumor model.

Mutatect MN-11 is a tumor line that can be grown subcutaneously in syngeneic C57BL/6 mice. The frequency of spontaneously arising mutants at the hypoxanthine phosphoribosyltransferase (Hprt) locus was observed to be elevated as a result of in vivo growth. The objective of the present study was to identify factors in the tumor microenvironment that might explain this increase in mutant frequency (MF). When tumors were examined histologically, neutrophils were found to be the predominant infiltrating cell type. Quantitative estimates of the number of neutrophils and MF of tumors in different animals revealed a statistically significant correlation (r = 0.63, P < 0.0001). Immunohistochemical analysis for inducible nitric oxide synthase (iNOS) demonstrated its presence, mainly in neutrophils. Biochemical analysis of tumor homogenates for nitric oxide synthase (NOS) activity indicated a statistically significant correlation with MF (r = 0.77, P < 0.0001). Nitrotyrosine was detected throughout the tumor immunohistochemically; both cytoplasmic and nuclear staining was seen. To increase the number of infiltrating neutrophils, tumors were injected with chemoattractant interleukin-8 and prostaglandin E2. This produced a statistically significant increase in neutrophil content (P = 0.005) and MF (P = 0.0002). As in control MN-11 tumors, neutrophil content and MF were strongly correlated (r = 0.63, P = 0. 003). Because neutrophils are a potential source of genotoxic reactive oxygen and/or nitrogen species, our results support the notion that these tumor-infiltrating cells may be mutagenic and contribute to the burden of genetic abnormalities associated with tumor progression.

Expression of serum- and glucocorticoid-regulated kinase (sgk) mRNA is up-regulated by GM-CSF and other proinflammatory mediators in human granulocytes.

Stimulation of human peripheral blood granulocytes with the proinflammatory cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), increases incorporation of [3H]uridine into RNA. We investigated the nature of the RNA synthesized under these conditions. Using transcription inhibitors, gel electrophoresis, and high-salt precipitation, it was concluded that as much as 90% of this radiolabeled RNA represents polymerase II transcripts. Differential display reverse transcription-polymerase chain reaction was used to identify and clone GM-CSF-responsive mRNAs. Serum- and glucocorticoid-regulated kinase (sgk) mRNA was identified that could be up-regulated 10- to 20-fold by > or =0. 1 ng/mL recombinant human GM-CSF. The 2.6-kb sgk mRNA was induced rapidly (within 30 min) by GM-CSF and remained at high levels for at least 12 h. Up-regulation was blocked completely by the transcription inhibitor, actinomycin D, but not by the translation inhibitor, cycloheximide, nor by the tyrosine kinase inhibitor, genistein. Up-regulation did not appear to be caused by enhanced mRNA stability. Other inflammatory mediators could also increase sgk mRNA levels (GM-CSF > > lipopolysaccharide > fMLP = tumor necrosis factor alpha). The function of sgk in granulocytes remains unknown.

Expression of interleukin-8 promotes neutrophil infiltration and genetic instability in mutatect tumors.

Neutrophils represent a potential source of genotoxic reactive oxygen and nitrogen species in the tumor microenvironment. Using Mutatect cell lines, which can form subcutaneous tumors in syngeneic C57BL/6 mice, we have previously established that the number of spontaneously infiltrating neutrophils correlates with the number of mutations at the hypoxanthine phosphoribosyltransferase (Hprt) locus. We now describe the properties of four lines that express different levels of the neutrophil chemokine, interleukin-8 (IL-8), from a tetracycline (TET)-responsive promoter. In a series involving 45 animals, IL-8-expressing lines produced tumors with a higher neutrophil content than the control line. Analysis of the 45 tumors revealed that the neutrophil level again strongly correlated with hprt mutant frequency (MF) (P<.0001, r=0.88). Administration of TET was effective in lowering the neutrophil content of low IL-8-expressing tumors, but not high IL-8-expressing tumors. Although the IL-8 transgene was stable in all lines in vitro, high IL-8-expressing lines completely lost the transgene in vivo whereas low IL-8-expressing lines showed no evidence of transgene instability. These results provide further evidence, based on the study of an endogenous gene (hprt) and an IL-8 transgene, that neutrophils may contribute to genetic instability in tumors.

Characterization of a polyclonal antibody to human thymidylate synthase suitable for the study of colorectal cancer specimens.

Measurement of thymidylate synthase (hTS) using immunohistochemical techniques has been reported in several clinical studies. However, its value as a prognostic indicator is still not clear. To pursue this, we have developed a new rabbit polyclonal antibody, hTS7.4. The antigen was recombinant hTS containing an N-terminal His(6)-tag. Antiserum hTS7.4 detected recombinant hTS by ELISA at a titer of 1:100,000. Western blot analysis of several human cell lines showed a single band of the expected 36-kD molecular size. HeLa cells treated with the TS inhibitor 5-FUdR showed the expected additional band corresponding to the ternary complex of hTS-dFUMP-reduced folate. hTS7.4 detected TS in bacterial, rat, mouse, and monkey cell extracts, and hTS8.3 (a closely related antiserum) immunoprecipitated a 36-kD [(35)S]-methionine-labeled protein from HeLa extracts. TS was detectable by indirect immunofluorescence in HeLa cells. Proliferating normal human fibroblasts in culture showed staining, but nonproliferating cells did not. Lymphocytes in the germinal center of human tonsil tissue, which are known to be proliferating, stained with hTS7.4 and also with monoclonal antibody TS106. TS may therefore be useful as an immunohistochemical marker of cell proliferation. Normal colon mucosa showed weak staining, whereas some colorectal cancer specimens stained very strongly with hTS7.4. A clinical study of colorectal cancer using this antibody is in progress. (J Histochem Cytochem 47:1563-1573, 1999)

A myeloperoxidase-specific assay based upon bromide-dependent chemiluminescence of luminol.

Measurement of myeloperoxidase (MPO; EC 1.11.1.7) activity is often used as a marker of neutrophil infiltration into tissues. However, most enzymatic assays for MPO are susceptible to interference from other peroxidases (including eosinophil peroxidase, EPX) and hemoproteins (such as hemoglobin and myoglobin) present in the tissues. In this report, we describe a bromide-dependent chemiluminescence (Br-CL) assay that uses luminol as a chemiluminescence probe. The assay can distinguish between MPO and nonspecific peroxidase reactions. The MPO-specific reaction is believed to proceed in two steps: (i) the enzymatic generation of hypobromous acid (HOBr) from KBr and H(2)O(2) at pH 5 and (ii) the spontaneous reaction of HOBr and H(2)O(2) with luminol to give a Br-CL signal. The assay is sufficiently sensitive to allow detection of MPO in <100 human neutrophils. Other peroxidases and hemoproteins do not interfere with the Br-CL signal. Although EPX can also oxidize bromide to generate HOBr, activities of MPO and EPX can be distinguished at different pHs. As a demonstration of the utility of the Br-CL assay, MPO activity was measured in murine tumors known to be infiltrated with neutrophils. A statistically significant correlation was seen between MPO activity and histological neutrophil counts in the tumors (r = 0.69, P < 0.01, n = 14). The assay should have wide application for measuring the neutrophil content of tissues.

Elimination of non-viable 6-thioguanine-sensitive T cells from viable T cells prior to PCR analysis.

The study of T cell clones at the genomic level is expanding our understanding of their role in diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). We have been carrying out genotypic analysis by PCR of hypoxanthine phosphoribosyltransferase (hprt) mutations in these cells. Mutant T cells in the population can be cloned on the basis of their resistance to the cytotoxic drug, 6-thioguanine-(6-TG). A difficulty is that the majority of primary human T cells are capable of only limited growth ex vivo, even in the presence of 'feeder' cells. PCR analysis of DNA from such clones is made difficult by the limited number of viable mutant (drug-resistant) T cells and the large number of dead (drug-sensitive) mononuclear cells and feeder cells. DNA from the 'dead' cells remains sufficiently intact for many weeks in culture and can represent a significant source of background in PCR analysis. Here we describe a method employing hypotonic shock and micrococcal nuclease that reliably eliminates non-viable 6-TG-sensitive cells, allowing the study of the hprt gene in < 200 T cells by PCR.

Low concentrations of the feverfew component parthenolide inhibit in vitro growth of tumor lines in a cytostatic fashion.

Parthenolide, a clinically useful agent and migraine prophylaxis principle from the medicinal plant, feverfew (Tanacetum parthenium), was tested on two tumor cell lines for its ability to inhibit cell growth. At concentrations above 5.0 microM and an exposure time of 24 h, parthenolide inhibited cell growth in an irreversible fashion. However, at lower concentrations, the effect was reversible; parthenolide acted in a cytostatic fashion over multiple cell generations for mouse fibrosarcoma (MN-11) and human lymphoma (TK6) cell lines. After 24 h exposure to 2.5 microM parthenolide, approx. 85% of cells were able to continue cell cycling on removal of the chemical, as demonstrated by labeling of S-phase cells with BrdU. In a clonogenic assay, colony formation was also unchanged by exposure to this concentration of parthenolide. No indication of cell synchronization could be found, as evidenced by the lack of appearance of a peak of mitotic figures when cells were examined at 1 h intervals for 10 h after drug removal. The mechanism of the reversible growth inhibition is uncertain.

Mutatect: a mouse tumour model for detecting radiation-induced mutations in vivo.

A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosphoribosyltransferase) locus is described. It is highly sensitive to detection of mutants induced by clastogenic agents such as ionizing radiation. MN-11 cells are grown as a subcutaneous tumour in C57BL/6 mice for a period of 2 weeks, during which time they can be exposed to mutagenic treatments. Cells taken from the animal are cultured ex vivo and 6-thioguanine (6-TG)-resistant mutant clones can be readily identified and scored. This model system may have special utility for detecting multi-locus deletion events (chromosomal mutations) induced by high LET forms of radiation that might be encountered in space.

HPRT- mutant T cells in the peripheral blood and synovial tissue of patients with rheumatoid arthritis.

OBJECTIVE: To investigate the frequency and characteristics of hprt- mutant T lymphocytes in the peripheral blood and synovium of rheumatoid arthritis (RA) patients compared with controls, and to correlate these findings with disease parameters. METHODS: An hprt- T cell assay was performed on blood and synovial samples from 93 RA patients, 8 osteoarthritis (OA) patients, and 19 control subjects. T cell clones were studied by flow cytometry and evaluated for fibronectin adhesion. RESULTS: RA patients showed a 5-fold increase in the frequency of mutant T cells in the peripheral blood compared with that in control peripheral blood, and a further 10-fold increase in the mutant T cell frequency in synovial tissue. In OA patients, the synovium also had a significantly higher frequency of hprt- mutant T cells compared with the peripheral blood, but at a lower level than in the rheumatoid synovium. RA peripheral blood mutant T cell clones displayed elevated fibronectin adhesion and beta1 integrin expression, similar to that observed in the RA synovial T cell lines. CONCLUSION: The origin of the mutated T cells in the peripheral blood of these patients appears to be the inflamed synovium of RA, and to a lesser extent, of OA, where the cells are exposed to a mitogenic and genotoxic environment.

Preliminary characterization of the protein encoded by human testis-enhanced gene transcript (TEGT).

TEGT is a conserved, widely expressed gene transcript of unknown function that has been studied previously only at the nucleic acid level. The deduced amino acid sequence predicts a highly hydrophobic, 26.5 kDa integral membrane protein with seven potential transmembrane domains. Little else is known about TEGT protein because of the lack of definitive homology to other known sequences and the absence of informative consensus motifs. The present report details a preliminary study of human TEGT (hTEGT) protein. (i) In vitro translation of hTEGT in reticulocyte lysates required the presence of microsomes for efficient synthesis, suggesting that hTEGT must target to the endoplasmic reticulum to be translated. Immunofluorescence of cells transiently expressing haemagglutinin-tagged hTEGT localized the protein mainly to the endoplasmic reticulum. The protein demonstrated no obvious post-translational modifications such as signal-peptide cleavage, N-linked glycosylation or O-linked glycosylation. (ii) Both hTEGT and haemagglutinin-tagged hTEGT appeared to retain partial secondary and tertiary structure in the presence of SDS. Both electrophoresed as a broad band or doublet with apparent molecular weights of 22-24.5 kDa on SDS-PAGE, aggregated either homotypically or heterotypically when boiled in SDS, and were toxic after 24 h when highly overexpressed in 293 T cells. These properties are believed to be caused by the protein's hydrophobicity. (iii) The protein appeared to associate strongly with other intracellular molecules since haemagglutinin-tagged hTEGT was extracted poorly from transiently transfected HeLa cells. Further study will be required to determine the cellular function of TEGT.

Mutagenicity and cytotoxicity of reactive oxygen and nitrogen species in the MN-11 murine tumor cell line.

There is increasing evidence that endogenously generated reactive oxygen (ROS) and reactive nitrogen (RNS) species at sites of inflammation and in tumors may be genotoxic. We have developed a murine tumor model (MN-11) in which mutations at the hypoxanthine phosphoribosyltransferase (HPRT) locus, arising both in vitro and in vivo, can be detected. In the present report, we describe an in vitro study of the ability of ROS and RNS to induce mutations in our model system. 137Cs radiation and radiomimetic drugs caused a dose-dependent increase in mutant frequency. At D0, radiation induced about 170 mutants per 10(5) viable cells, compared to 50 and 95 for streptonigrin and bleomycin, respectively. H2O2 induced a lower frequency of mutants, 20-30 per 10(5), for enzymatically generated or bolus, respectively. For the following treatments, mutant frequency at 50% survival is shown. Incubation with human granulocytes induced a low frequency of mutants (about 15 per 10(5)). RNS was tested using a series of NO-donating drugs. Spermine/NO. induced cytotoxicity but no mutants while S-nitroso-N-acetylpenicillamine induced a low level, 10 per 10(5). Both release nitrogen monoxide spontaneously, with a t1/2 < 3 h. Glyceryl trinitrate and sodium nitroprusside are two drugs that were slowly metabolized by MN-11 cells (> 12 h). Glyceryl trinitrate induced about 20 per 10(5) while nitroprusside induced 50 per 10(5). Our results indicate that RNS can readily induce mutations detectable in MN-11 cells. At equicytotoxic doses, the induced mutant frequency varied considerably for different drugs, suggesting that different states of nitrogen monoxide (such as NO+ or NO.) may be generated and these may vary in their mutagenic/cytotoxic potential.

Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes.

Phorbol ester treatment of granulocytes triggers release of superoxide (O2.-) and a concomitant burst of DNA strand breaks. The relationship between the amount of O2.- and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O2.- generated over a 40-min period by 1 x 10(6) granulocytes/mL, a discontinuous "10-min pulse" method employing cytochrome c was used; 140 nmol O2.- per 1 x 10(6) cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O2.- released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1-10 mM) and staurosporine (2-10 nM) both inhibited O2.- production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O2.-. Zinc chloride (50-200 microM) inhibited both O2.- and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O2.- production more effectively than it inhibited DNA breaks. O2.- dismutes to H2O2, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H2O2 had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O2.- or H2O2 in PMA-stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway.

Receptor-mediated and protein kinase-dependent growth enhancement of primary human fibroblasts by platelet activating factor.

Chronic inflammation is a recognized risk factor for human cancer, but the causal mechanisms are poorly understood. We previously demonstrated that platelet activating factor (PAF) can induce alterations in the in vitro growth properties of primary rat fibroblasts. In the study reported here, exposure of primary human skin fibroblasts to PAF for 1 h in serum-free medium was shown to cause sustained proliferation over 50 d in medium containing low serum and anchorage-independent growth in soft agarose. Both properties could be inhibited by pretreatment with a PAF receptor antagonist, CV3988 (10 microM); a tyrosine-kinase inhibitor, genistein (1 microgram/mL); or a protein kinase C (PKC) inhibitor, staurosporine (50 nM) but not with a cyclooxygenase inhibitor, indomethacin (200 nM-20 microM). PAF had no effect on doubling time, saturation density, or cell viability under normal monolayer growth conditions in complete medium. Treatment with lyso-PAF, an inactive metabolite of PAF, had no effect in either of the assays. Control and PAF-induced cell proliferation in low-serum medium was inhibited by PAF receptor antagonists present during the extended growth period. The presence of PAF receptor mRNA in human skin fibroblasts was demonstrated by reverse transcriptase-polymerase chain reaction. The presence of a functional receptor was indicated by an early (2 min) transient increase in PKC activity and an increase in fos mRNA after PAF treatment. PAF-induced PKC activity was blocked by pretreatment with either staurosporine (50 nM) or CV3988 (1 microM). These results suggest that PAF is a mitogenic factor that contributes to the known increase in risk of malignancy associated with chronic inflammatory conditions.

Hprt mutants in a transplantable murine tumour arise more frequently in vivo than in vitro.

A model system was developed to allow investigation of the frequency at which clastogenic and/or mutagenic events occur in situ in a transplantable murine fibrosarcoma tumour (MC1A-C1) compared with in vitro culture. The marker selected for detecting these events was the X-linked hprt (hypoxanthine-guanine phosphoribosyltransferase) gene. We found that the hprt gene in MC1A-C1 was not suitable for this purpose, most likely because multiple active copies were present. To circumvent the problem, HPRT- [6-thioguanine (6-TG)-resistant] clones were isolated by inactivating all hprt genes with methylnitrosourea. Spontaneous revertants to hypoxanthine/aminopterin/thymidine resistance (HATR) were isolated and found to be approximately 1000 times more sensitive than the parental tumour to induction of 6-TGR mutants by cobalt-60 gamma-rays. This sensitivity is expected for a heterozygous marker, these revertants may therefore possess only one functional hprt locus but two or more active X chromosomes. A clone with a stable hprt gene was identified and a neo gene was introduced. The resulting cell line (MN-11) could be grown as a subcutaneous tumour in syngeneic C57BL/6 animals. The frequency of mutations arising in vivo in the marker hprt gene could be estimated by culturing explanted tumour cells in the presence of 6-TG, using G418 selection to distinguish tumour from host cells. The frequency of mutants in MN-11 cells grown as tumours was found to be 3.4-fold higher than in tissue culture for an equivalent period of time. These data provide the first direct evidence for the existence of mutagenic factors in a tumour environment that might contribute to tumour progression.