H. Chaim Birnboim

Cutting Edge: MHC Class II-Restricted Peptides Containing the Inflammation-Associated Marker 3-Nitrotyrosine Evade Central Tolerance and Elicit a Robust Cell-Mediated Immune Response

Nitrotyrosine is widely recognized as a surrogate marker of up-regulated inducible NO synthase expression at sites of inflammation. However, the potential immunogenicity of autologous proteins containing nitrotyrosine has not previously been investigated. Herein, we used the I-EK-restricted T cell epitope of pigeon/moth cytochrome c (PCC/MCC88–103) to assess the ability of T cells to recognize ligands containing nitrotyrosine. Substitution of the single tyrosine (Y97) in PCC/MCC88–103 with nitrotyrosine abrogates recognition by the MCC88–103-specific T cell hybridoma 2B4. CBA (H2K) mice immunized with MCC88–103 or nitrated MCC88–103 peptides produce T cell responses that are mutually exclusive. Transgenic mice that constitutively express PCC under the control of an MHC class I promoter are tolerant toward immunization with MCC88–103, but exhibited a robust immune response against nitrated MCC88–103. Analysis of T cell hybridomas specific for nitrated-MCC88–103 indicated that subtle differences in TCR VDJ gene usage are sufficient to allow nitrotyrosine-specific T cells to escape the processes of central tolerance.

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.

AmpFlSTR Profiler Plus and AmpFlSTR COfiler analysis of tissues stored in GenoFix, a new tissue preservation solution for mass disaster DNA identification.

A preliminary study was conducted to assess the capability of a new alcohol-based tissue fixative, GenoFix, to preserve DNA from biopsy tissues stored at room temperature and/or -20 degrees C in a freezer, for subsequent short tandem repeat (STR) DNA typing analysis. Fresh human smooth muscle samples were stored at room temperature in GenoFix for one month and up to one year and seven months before being processed using the megaplex STR systems, AmpFlSTR Profiler Plus and AmpFlSTR COfiler. Alternatively, muscle tissues in GenoFix were placed at -20 degrees C in a freezer for up to 3 1/2 years following two to three months in the fixative at room temperature. DNA analysis was also carried out on tissues stored in GenoFix for one month at room temperature and subsequently paraffin-embedded and stored at room temperature for four years. The AmpFlSTR Profiler Plus and AmpFlSTR COfiler STR profiles produced, using DNA extracted from all fixed tissue samples, were of very good quality. The fluorescent signals were well balanced across the nine STR loci or six loci comprised in the megaplexes surveyed and profiles showed no differences with those observed for the control blood of the respective donor patients. Continuous exposure to GenoFix at room temperature (up to one year and seven months) did not compromise the STR typing analysis of the fixed tissues. No adverse effects were noted on the STR typeability of tissues fixed with GenoFix and stored at -20 degrees C in a freezer for up to 3 1/2 years. STR profiles generated from the paraffin-embedded tissues fixed in GenoFix were of excellent quality. This preliminary study suggests that GenoFix can be used to store tissue samples at room temperature for up to one year and seven months or at -20 degrees C in a freezer for longer storage (up to 3 1/2 years). This new and odorless tissue fixative promotes tissue and DNA preservation in a very effective manner and as such may prove useful in criminal investigations or mass disaster identifications carried out in remote locations and in which a small or large number of tissue samples are collected for further analyses.

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.

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.

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 μM); a tyrosine‐kinase inhibitor, genistein (1 μg/mL); or a protein kinase C (PKC) inhibitor, staurosporine (50 nM) but not with a cyclooxygenase inhibitor, indomethacin (200 nM–20 μM). 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 μM). These results suggest that PAF is a mitogenic factor that contributes to the known increase in risk of malignancy associated with chronic inflammatory conditions. Mol. Carcinog. 20:366–375, 1997.

Nuclear Expression of Thymidylate Synthase in Colorectal Cancer Cell Lines and Clinical Samples

Thymidylate synthase (TS) [TYMS; OMIM reference number (188, 350)] is normally considered to be a cytoplasmic enzyme. However, a few reports have suggested it may also be present in the nucleus. To explore this in more detail, we used a highly specific polyclonal antibody to TS and a combination of techniques, including immunocytochemistry, confocal microscopy, cell fractionation, and Western blotting. We developed cell line HeLa-55, a HeLa derivative that grossly overexpresses TS. Although the vast majority of TS was in the cytoplasm, some TS also was seen in the nucleus. TS in parental HeLa cells and in normal human fibroblasts was seen exclusively in the cytoplasm. HeLa-55 cells exposed to 5-fluorodeoxyuridine were fractionated and examined by Western blotting. Interestingly, both free TS and the ternary complex of TS were seen in the cytoplasmic fraction but only free TS was detected in the nuclear fraction. Amongst different cell lines examined, HCT-15 and normal fibroblasts showed no nuclear TS, HCC-2998 and SW-620 showed a small amount of nuclear TS, and HT-29, RKO, and HCT-116 showed a strong nuclear TS signal. Nuclear staining was clearly evident in some clinical colorectal specimens, both normal and malignant. This staining was definitively shown to be TS by competition with recombinant TS protein. A putative leucine-rich nuclear export sequence was identified but its function could not be confirmed. We conclude that small amounts of TS protein is present in the nucleus of some cell types but further work is needed to determine the significance of this observation.

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

Phorbol ester treatment of granulocytes triggers release of superoxide (O  2 − ) and a concomitant burst of DNA strand breaks. The relationship between the amount of O  2 − and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O  2 − generated over a 40‐min period by 1 × 106 granulocytes/mL, a discontinuous “10‐min pulse” method employing cytochrome c was used; 140 nmol O  2 − per 1 × 106 cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O  2 − released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1–10 mM) and staurosporine (2–10 nM) both inhibited O  2 − production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O  2 − . Zinc chloride (50–200 μM) inhibited both O  2 − and DNA breaks, approximately equally. Dinophysistoxin‐1 (okadaic acid) inhibited O  2 − production more effectively than it inhibited DNA breaks. O  2 − 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 O  2 − 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. J. Cell. Biochem. 66:219–228, 1997.

Potentiation of retinoic acid-induced U-937 differentiation into respiratory burst-competent cells by nitric oxide donors.

All-trans retinoic acid (tretinoin) is a known inducer of differentiation of the human monoblastic cell line, U-937. We now report that the ability of retinoic acid (RA) to induce differentiation of U-937 cells into cells possessing respiratory burst activity is enhanced by the known nitric oxide-donating drugs glyceryl trinitrate, molsidomine and CAS 936, and by tetranitromethane in combination with cysteine. RA alone was a strong inducer of U-937 differentiation as indicated by the following responses to 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation: (1) increase in the percentage of cells staining with nitroblue tetrazolium (NBT); (2) increase in the total amount of formazan (the product of NBT reduction by O2-.) as determined spectrophotometrically; (3) increase in hexose monophosphate shunt (HMPS) activity as assessed by [14C]CO2 released from D-[1-14C]glucose. RA was also able to increase mRNA levels for two respiratory burst-related genes and for glucose-6-phosphate dehydrogenase (G6PD), an HMPS enzyme. Other indications of differentiation were reduced cell proliferation, increased adherence and altered nuclear morphology. The observed increase in formazan production and HMPS activity and the reduction of cell proliferation due to RA were augmented by co-treatment with either glyceryl trinitrate, molsidomine, CAS 936 or tetranitromethane plus cysteine. Glyceryl trinitrate alone increased HMPS activity and G6PD mRNA levels and also reduced cell proliferation. Glyceryl trinitrate, molsidomine and CAS 936 are presumed to release nitric oxide and increase intracellular cGMP levels by stimulation of soluble guanylate cyclase. The mechanism of action of tetranitromethane is less certain, although it may also generate reactive nitrogen intermediates. These data suggest that a NO./cGMP pathway may augment a retinoic acid-mediated pathway to enhance maturation of U-937 cells with respect to the respiratory burst. Glyceryl trinitrate may act additionally by another pathway.

EGFR expression variance in paired colorectal cancer primary and metastatic tumors

Background: Previous studies indicate that drugs targeting the Epidermal Growth Factor Receptor (EGFR) signaling pathways can induce objective responses, prolong time to progression and improve survival of patients with metastatic colorectal cancer (mCRC). EGFR expression in the primary tumour may not predict response to these agents and data is conflicting regarding the correlation of EGFR expression in the primary tumour with the metastatic site. In other tumour sites, the presence of EGFR mutations was associated with efficacy in a subset of patients. Objectives: The goal of this study is to correlate tumour EGFR expression between primary and liver metastatic sites, and to assess the mutational status in the EGFR kinase domain. Methods: This is a single center retrospective study of patients who underwent surgical resection of CRC, for whom paired paraffin-embedded tissue blocks of primary tumours and resected liver metastases were available. EGFR immunostaining and mutation analyses were preformed. Results: Fifty eight paired colorectal primaries and metastases were available for analysis. EGFR was detectable in 96.6% of the primary samples and in 89.7% of the metastatic samples. Perfect concordance in the intensity score between the primary and the metastases was found in 46.5% of the cases. While individual pairs were poorly concordant for intensity, the proportion of primaries with intense staining was similar to the proportion with intense staining in the metastatic samples. Overall survival did not correlate with either EGFR expression in the primary tumour, or with EGFR expression in the metastasis. There were 2 cases with mutations in the EGFR kinase domain. Both mutations were found in exon21 C>T. Conclusions: In this analysis, EGFR expression in the primary tumor site was not predictive of its level in the metastasis. EGFR expression levels in the primaries and in the metastases do not appear to be useful prognostic markers.