RAD51 gene polymorphisms and sporadic colorectal cancer risk in Poland.

BACKGROUND: DNA repair processes play an important role in protection against carcinogenic factors. Mutations in DNA repair genes, which code proteins engaged in repair processes, may lead to carcinogenesis and among others also to colorectal cancer (CRC) development. The genetic variability in RAD51 may contribute to the appearance and progression of various cancers including CRC. The aim of the study was to compare the distribution of genotypes of RAD51 135G>C and 172G>T polymorphism between colorectal cancer patients and controls. MATERIAL AND METHODS: Both polymorphisms were evaluated by PCR-RFLP methods in colorectal tissue of 320 colorectal cancer subjects and 320 healthy subjects who served as controls. RESULTS: In the present work we demonstrated a significant positive association between the RAD51 C/C genotype and colorectal carcinoma. Variant 135C allele of RAD51 increased the cancer risk. However, we did not observe any relationship between each polymorphism and colorectal cancer progression assessed by node metastasis, tumour size and Dukes' stage. CONCLUSIONS: Our results suggest that variant genotypes of the 135G>C of RAD51 polymorphism may be positively associated with colorectal carcinoma in the Polish population. Further studies conducted on a larger group are required to clarify this point.

Platelet activation patterns are different in mouse models of diabetes and chronic inhibition of nitric oxide synthesis.

Currently, there are several animal models of diabetes mellitus and hypertension, but relatively little is known about blood platelet function in these models. The aim of this work was to characterise and compare platelet reactivity and activation in db/db mice (mouse model of diabetes) and mice receiving L-NAME (model of chronic inhibition of NO synthesis), using various platelet function assays. We found higher platelet activation (circulating resting platelets) in db/db mice than in db/+heterozygotes, as evidenced by elevated expressions of CD62P and CD40L and a lower expression of CD42b. The expression of COX-1 was significantly increased, and the phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser(157) significantly reduced in platelets from db/db mice. Similarly, we observed platelet hyperreactivity in db/db mice following the in vitro responses to 20µg/ml collagen (reflected by increased expressions of CD62P and CD40L, and reduced CD42b), 20µM ADP (reduced CD42b) and lower concentrations of thrombin (0.025 U/ml) (increased CD62P, JON/A, bound vWF, and bound fibrinogen). Otherwise, platelet hyporeactivity was revealed for higher thrombin (0.25 U/ml) (reduced CD62P and bound vWF), while hyperreactivity occurred for CD40L and bound Fg in db/db mice compared to non-diabetic control, db/+. Plasma levels of sCD40L, but not of sCD62P, were increased in db/db mice; also plasma TXB2 concentrations were over 3.5-fold higher in this group than in the heterozygous db/+mice (P<0.01). In contrast, in the mice administered with L-NAME, no statistical differences in expressions of platelet activation markers were found between mice supplemented with L-NAME and controls. Likewise, the TXB2 level did not differ between L-NAME mice and controls, but L-NAME mice had significantly higher plasma levels of sCD62P and sCD40L than controls. In conclusion, these two studied models differ in the overall picture of blood platelet activation and reactivity, as they demonstrated opposite time sequence patterns of platelet activation in circulating blood. More generally, our study provides another argument for the opinion that multiparametric analysis of platelet function offers a much better tool for investigation and minimizes the likelihood of artefacts.

Can the antiplatelet effects of cangrelor be reliably studied in mice under in vivo and in vitro conditions using flow cytometry?

BACKGROUND: The effects of blood platelet inhibitors are often not quite equivalent under in vivo and in vitro conditions. Amongst various models of human pathology using laboratory animals, mice offer several benefits that make them convenient tools for studying the putative therapeutic value of various compounds. However, despite its advantages, the mouse model has methodological limitations concerning the small amount of blood available and technical difficulties with its collection. Among the variety of available methods used to study blood platelet activation and/or reactivity, flow cytometry seems an attractive technique that largely minimizes the constraints of using small rodents and enables outcomes of laboratory research to be transferred successfully to clinical practice. In this study we aimed at a critical evaluation of the optimal discriminative flow cytometric protocol, useful for reliable studies of the effect of cangrelor, a P2Y12 receptor antagonist, on mouse platelets under in vitro and in vivo conditions. METHODS: Blood samples were drawn from two-month-old female BALB/c mice. Protocols differing in methods of anesthesia, blood withdrawal, anticoagulation, gating antibodies, blood preparation and fixation were tested to optimize the one best suited to discrimination between resting and activated platelets. The antiplatelet capabilities of cangrelor were tested in vitro (140 µM in whole blood) and in vivo (7.8 mg/kg b.w. administered once, directly into the bloodstream through the vena cava of the anesthetized animal, 15 min prior to blood withdrawal). Expressions of P-selectin, activated α(IIb)ß3 complex and GPIba were monitored using two-color flow cytometry. RESULTS: "Washed blood" anticoagulated with low molecular weight heparin demonstrated the best discrimination between circulating (resting) platelets and upon their in vitro response to thrombin, collagen or ADP in freshly-stained unfixed cell suspensions. Cangrelor inhibited the expression of the active form of the integrin a(IIb)ß3 to approximately the same extent under in vitro and in vivo conditions (84.5 ± 7.7% vs. 75.4 ± 19.5% for the in vitro and in vivo approaches, respectively, n.s.). CONCLUSIONS: The agreement between the in vivo and in vitro approaches with respect to cangrelor-inhibited hallmarks of blood platelet activation and reactivity supports our proposal that flow cytometry is useful and reliable for determining the effects of antiplatelet agents on the activation of circulating platelets in the mouse model, as well as the in vitro response of platelets to agonists.