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Vitamin E in foodstuffs is a mixture of tocopherols. In mouse Mutatect tumors, a model designed to detect DNA mutations, the hypoxanthine phosphoribosyltransferase (Hprt) gene mutation frequency is associated with the number of tumor-infiltrating neutrophils and both are markedly decreased in mice fed high levels of [alpha]-tocopherol. Dietary [alpha]-tocopherol is also associated with a decrease in neutrophil-associated loss of an inter-leukin 8 (IL-8)-expressing transgene in this tumor model. We examined Hprt gene mutation frequency (expressed as the number of 6-thioguanine-resistant colonies per [10.sup.5] clonable tumor cells), IL-8 transgene loss, and myeloperoxidase activity (an indirect measure of neutrophil number) in tumors from Mutatect mice fed diets supplemented with various concentrations of D-[alpha]-tocopherol acetate and/or D-[gamma]-tocopherol acetate or neither tocopherol for 4 weeks. Hprt gene mutation frequency and myeloperoxidase activity were statistically significantly lower in tumor cells from mice fed [alpha]-tocopherol at 50 or 100 mg/kg body weight per day than in tumor cells from mice fed 0 mg/kg body weight per day [alpha]-tocopherol (P

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 [is less than] .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. [J Natl Cancer Inst 2000;92: 1429-33]

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.

Nitric oxide-derived reactive species have been implicated in many disorders. Protein nitrotyrosine is often used as a stable marker of these reactive species. Using immunohistochemistry, we have previously detected nitrotyrosine in murine Mutatect tumors, where neutrophils are the principal source of nitric oxide. We now report on the identification of several prominent nitrotyrosine-containing proteins. Using Western blot analysis, nitrotyrosine in higher molecular mass proteins (>20 kDa) was detected in tumors containing a high number of neutrophils but not in tumors with fewer neutrophils. Staining for nitrotyrosine was consistently seen in low molecular mass proteins (≤15 kDa), regardless of the level of neutrophils. Protein nitrotyrosine was not seen in Mutatect cells growing in vitro. Treatment with nitric oxide donors produced nitration of ≤15-kDa proteins, but only after extended periods. These small proteins, both from tumors and cultured cells, were identified by mass spectrometry to be histones. Only a subset of tyrosine residues was nitrated. Selective nitration may reflect differential accessibility of different tyrosine residues and the influence of neighboring residues within the nucleosome. The prominence of histone nitration may reflect its relative stability, making this post-translational modification a potentially useful marker of extended exposure of cells or tissues to nitric oxide-derived reactive species.

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 hprtmutations 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 Hprtmutant clones from patients is appreciably elevated compared to that of control subjects. Large‐scale hprtdeletions (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. Environ. Mol. Mutagen. 38:261–267, 2001. © 2001 Wiley‐Liss, Inc.

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 vivogrowth. 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.