In this case-control study, we modified the cytokinesis-block micronucleus (CBMN) assay, an established biomarker for genomic instability, to evaluate susceptibility to the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by measuring the frequency of NNK-induced chromosomal damage endpoints (micronuclei, nucleoplasmic bridges, and nuclear buds) per 1,000 binucleated lymphocytes. Spontaneous and NNK-induced chromosomal damage were significantly higher in lung cancer patients compared with controls. Forty-seven percent of cases (versus 12% of controls) had z4 spontaneous micronuclei, 66% of cases (and no controls) had z4 spontaneous nucleoplasmic bridges, and 25% of cases (versus 5% of controls) had z1 spontaneous nuclear bud (P < 0.001). Similarly, 40% of cases (versus 6% of the controls) had z5 NNK-induced micronuclei, 89% of cases (and no controls) had z6 induced nucleoplasmic bridges, and 23% of cases (versus 2% of controls) had z2 induced nuclear buds (P < 0.001). When analyzed on a continuous scale, spontaneous micronuclei, nucleoplasmic bridges, and nuclear buds were associated with 2-, 29-, and 6-fold increases in cancer risk, respectively. Similarly, NNKinduced risks were 2.3-, 45.5-, and 10-fold, respectively. We evaluated the use of CBMN assay to predict cancer risk based on the numbers of micronuclei, nucleoplasmic bridges, and nuclear buds defined by percentile cut points in controls. Probabilities of being a cancer patient were 96%, 98%, and 100% when using the 95th percentiles of spontaneous and NNK-induced micronuclei, nucleoplasmic bridges, and nuclear buds, respectively. Our study indicates that the CBMN assay is extremely sensitive to NNK-induced genetic damage and may serve as a strong predictor of lung cancer risk. (Cancer Res 2006; 66(12): 6449-56)
The multi-endpoint cytokinesis-blocked micronucleus assay is used for assessing chromosome aberrations. We have recently reported that this assay is extremely sensitive to genetic damage caused by the tobaccospecific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and that the binucleated cells with micronuclei, nucleoplasmic bridges, and nuclear buds in lymphocytes (chromosome damage endpoints measured by the assay) are strong predictors of lung cancer risk. In the current study, we refined our analysis to include toxicity endpoints (micronuclei in mononucleated cells, apoptosis, necrosis, and nuclear division index) to investigate the benefit of including these variables on improving the predictive value of the assay. Baseline and NNK-induced micronuclei in mononucleated cells were significantly higher in patients (n = 139) than controls (n = 130; P < 0.001). Baseline apoptosis was higher among cases; however, the controls showed a significant higher fold increase in NNK-induced apoptosis compared with baseline (P < 0.001). Principal components analysis was used to derive a summary measure for all endpoints and calculate the positive predictive value (PPV) and negative predictive value (NPV) for disease status. First principal component for NNK-induced chromosome damage endpoints (binucleated cells with micronuclei, nucleoplasmic bridges, and nuclear buds) had an area under the curve = 97.9 (95% confidence interval, 95.9-99.0), PPV = 94.8, and NPV = 92.6. The discriminatory power improved when micronuclei in mononucleated cells were included: area under the curve = 99.1 (95% confidence interval, 97.9-100.0), PPV = 98.7 and NPV = 95.6. The simplicity, rapidity, and sensitivity of the assay together with potential for automation make it a valuable tool for screening and prioritizing potential cases for intensive screening. (Cancer Epidemiol Biomarkers Prev 2008;17(5):1111 -9)
Canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The cells were grown under adherent conditions. The growth, cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Ten female Balb/SCID mice were inoculated with IPC-366 cells to assess their tumorigenicity and metastatic potential. Chromosome aberration test and Karyotype revealed the presence of structural aberration, numerical and neutral rearrangements, demonstrating a chromosomal instability. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon that has been related to s vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2, E-cadherin, overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). At 2 weeks after inoculating the IPC-366 cells, a tumor mass was found in 100 % of mice. At 4 weeks metastases in lung and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative study of both IMC and IBC.
Polymorphisms in DNA-repair genes could contribute to the interindividual differences in cancer susceptibility in smokers. By reducing DNA-repair capacity, these polymorphisms may influence the net level of smoking-induced genetic damage significantly, a critical step in the cascade of events leading to cancer. In this biomonitoring study, we examined the relationship between polymorphisms in the DNA-repair gene XPD/ERCC2 and genetic damage. We tested the hypothesis that coding polymorphisms in XPD/ERCC2 limit DNA-repair efficiency in humans leading to increased frequencies of chromosome aberration (CA) in their lymphocytes. We also used the mutagen-sensitivity assay, with the tobacco-specific nitrosamine NNK as a model mutagen, to determine whether lymphocytes from individuals with the variant XPD alleles are more sensitive to this tobacco-specific carcinogen. We calculated odds ratios (ORs) as estimates of relative risk of increased frequencies of CA associated with two XPD polymorphisms (Asp312Asn in exon 10 and Lys751Gln in exon 23). We observed a 2.57-fold (95% confidence limit [CL] = 0.88-7.50; P = 0.10) increase in risk of elevated in vivo frequencies of CA associated with the variant 312Asn allele in the total population. The relative risk was more pronounced in smokers (OR = 4.67; 95% CL = 1.04-20.90; P = 0.04) and in all subjects >48 years old (OR = 7.33; 95% CL = 1.53-35.10; P = 0.01). Similarly, elevations in NNK-induced aberrations were significantly associated with the 312Asn allele (OR = 3.69; 95% CL = 1.29-10.56; P = 0.02). The risk was higher in smokers (OR = 4.62; 95% CL = 1.14-18.70; P = 0.04) and in subjects >48 years old (OR = 5.76; 95% CL = 1.30-25.41; P = 0.03). No significant effect was observed with the 715Gln variant allele in relation to either in vivo or NNK-induced CA. These data suggest that the Asp312Asn polymorphism may alter the phenotype of the XPD protein, resulting in reduced DNA-repair capacity.
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