[Study on the association between CYP24A1 genetic polymorphisms and risks related to postmenopausal breast cancer].

Objective: To evaluate the associations between CYP24A1 genetic polymorphisms and related risks on breast cancer among postmenopausal women. Methods: We carried out a population-based case-control study to include 1 134 postmenopausal women (589 cases and 545 controls) from Wuxi, Jiangsu province and to explore the association between CYP24A1 polymorphisms and related risks on breast cancer. Seven CYP24A1 variants (rs2209314, rs2585428, rs2762941, rs3787555, rs4909959, rs912505 and rs927650) were genotyped by Sequenom MassARRAY platform. Logistic regression method was used to estimate the CYP24A1 genetic variants and susceptibility of breast cancer. Loci-loci interactions were evaluated by a generalized multifactor dimensionality reduction (GMDR) method. Results: Result showed that rs2209314, rs2585428, rs2762941, rs3787555, rs4909959, rs912505 and rs927650 of CYP24A1 were not associated with breast cancer under the codominant, dominant, recessive or additive models. Among the population with <80 cm waist circumstance, rs2585428 was associated with the reduced risks on breast cancer (OR=0.64, 95%CI: 0.42-0.96). Similar negative association was observed for rs3787555 (OR=0.58, 95%CI: 0.38-0.87). The genotypes of rs2585428, rs3787555 and rs4909959 showed significant interactions with waist circumstance on the risk of breast cancer. Also, rs2209314, rs3787555 and rs912505 in CYP24A1 could alter the risk of breast cancer by way of loci-loci interaction. Conclusion: CYP24A1 variants rs2585428 and rs3787555 were associated with risks of susceptibility on breast cancer, among postmenopausal women.

Ultrastructure of intestinal mucosa in diarrhea-predominant irritable bowel syndrome.

OBJECTIVES: Impaired intestinal barrier function has been demonstrated in the pathophysiology of diarrhea-predominant irritable bowel syndrome (IBS-D). This study aimed to describe the intestinal ultrastructural findings in the intestinal mucosal layer of IBS-D patients. METHODS: In total, 10 healthy controls and 10 IBS-D patients were analyzed in this study. The mucosa of each patient's rectosigmoid colon was first assessed by confocal laser endomicroscopy (CLE); next, biopsied specimens of these sites were obtained. Intestinal tissues of IBS-D patients and healthy volunteers were examined to observe cellular changes by transmission electron microscopy (TEM). RESULTS: CLE showed no visible epithelial damage or inflammatory changes in the colonic mucosa of IBS-D compared with healthy volunteers. On transmission electron microscopic examination, patients with IBS-D displayed a larger apical intercellular distance with a higher proportion of dilated (>20 nm) intercellular junctional complexes, which was indicative of impaired mucosal integrity. In addition, microvillus exfoliation, extracellular vesicle as well as increased presence of multivesicular bodies were visible in IBS-D patients. Single epithelial cells appeared necrotic, as characterized by cytoplasmic vacuolization, cytoplasmic swelling, and presence of autolysosome. A significant association between bowel habit, frequency of abdominal pain, and enlarged intercellular distance was found. CONCLUSION: This study showed ultrastructural alterations in the architecture of intestinal epithelial cells and intercellular junctional complexes in IBS-D patients, potentially representing a pathophysiological mechanism in IBS-D.

Expression of CD27, CD28 and IL-17A in peripheral blood from patients with colorectal carcinoma.

OBJECTIVE: To compare the different expressions of CD27, CD28, IL-17A, IFN-γ and TNF-α in the peripheral blood sampled from patients with colorectal carcinoma and healthy volunteers. PATIENTS AND METHODS: Vδ2 T cells were isolated from the peripheral blood mononuclear cells (PBMCs) of patients with the colorectal carcinoma (CRC, n = 30) and healthy controls (HC, n = 21). The proportion of CD27, CD28, IL-17A, IFN-γ and TNF-α of Vδ2 T cells was detected by the flow cytometry. RESULTS: We found that the proportion of IL-17A of Vδ2 T cells in PBMCs was higher in the CRC vs. the HC group (p < 0.05). A significant positive correlation was observed between the expression of IFN-γ and TNF-α of Vδ2 T cells. In the CRC patients, the proportions of IL-17A of CD27- Vδ2 T cells and CD28+ Vδ2 T cells were higher than those of CD27+ Vδ2 T cells and CD28- Vδ2 T cells, whereas the expression of IFN-γ and TNF-α of CD27-Vδ2 T cells was lower than that of CD27+ Vδ2 T cells. CONCLUSIONS: Vδ2 T cells from PBMCs had higher expression of IL-17A in CRC patients than that in the HC group. The expression of IFN-γ and TNF-α of Vδ2 T cells from PBMCs was positively correlated. The cytokine profiles of peripheral Vδ2 T cells were likely determined by a CD27 and CD28 involving mechanism.

Down-regulation of 67LR reduces the migratory activity of human glioma cells in vitro.

OBJECTIVES: Glioma is the most common brain tumor in central nervous system. Traditional therapies are not effective to cure this disease. Experimental evidence indicates that the 67 kDa elastin-laminin receptor (67LR) subunit is a high-affinity non-integrin laminin-binding protein that is over-expressed on the tumor cell surface in a variety of human carcinomas, and directly correlates with a higher proliferation rate of malignant cells and tendency to metastasize. However, little is known of the expression and function of 67LR in glioma cells. METHODS: In this study, we estimated whether 67LR was constitutively over-expressed in high-grade astrocytomas by immunohistochemical staining and Western blotting, and investigated the role of a low level of 67LR expression in glioma cell line-U251 by constructing an interfering RNA expression plasmid. RESULTS: The results showed that the 67LR had an enhanced over-expression in high-grade astrocytomas against normal brain tissues samples, and that the migratory activity of glioma cells was reduced after the down-regulation of the 67LR gene by RNAi. DISCUSSION: It was hypothesized that a low level of 67LR expression could reduce migratory activity of glioma cells, which further proved that 67LR played an important role in glioma invasion by mediating tumor cell functions leading to sarcomata. This study provided a new alternative to gene therapy for glioma treatment.

Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent.

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT --> TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10(-6), Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.

N-(2-chloroethyl)-N-nitrosourea tethered to lexitropsin induces minor groove lesions at the p53 cDNA that are more cytotoxic than mutagenic.

Many different N-chloroethyl-N-nitrosourea (CENU) derivatives have been synthesized in an attempt to minimize carcinogenic activity while favoring antineoplastic activity. CENU derivatives linked to the dipeptide lexitropsin (lex) showed significant changes in groove- and sequence-selective DNA alkylation inducing thermolabile N3-alkyladenines (N3-Alkyl-As) at lex equilibrium binding sites. CENU-lex sequence specificity for DNA alkylation was determined using 32P-end-labeled restriction fragments of the p53 cDNA. The adducted sites were converted into single-strand breaks by sequential heating at neutral pH and exposure to piperidine. To establish the mutagenic and lethal properties of CENU-lex-specific lesions, a yeast expression vector harboring a human wild-type p53 cDNA was treated in vitro with CENU-lex and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutants were isolated from independent ade- transformants. The results revealed that: (a) CENU-lex preferentially induces N3-Alkyl-A at specific lex equilibrium binding sites, the formations of which are strongly inhibited by distamycin; (b) reactivity toward Gs is still present, albeit to a lesser extent when compared to N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea and to CENU; (c) 91% of the 49 CENU-lex p53 mutations (45 of 49) were bp substitutions, 29 of which were GC-->AT transitions, mainly at 5' purine G sites; (d) all AT-targeted mutations but one were AT-->TA transversions; (e) the distribution of the CENU-lex mutations along the p53 cDNA was not random, with position 273 (codon 91), where only GC-->AT transitions were observed, being a real (n = 3, P < 0.0002) CENU-lex mutation hot spot; and (f) a shift in DNA alkylation sites between lesion spectra induced by CENU-lex and N-(2-chloroethyl-N-cyclohexyl-N-nitrosourea was associated with an increased lethality and a decreased mutagenicity, whereas no dramatic change in mutational specificity was observed. Hence, it is tempting to conclude that, in this experimental system, N3-Alkyl-A is more lethal than mutagenic, whereas O6-alkylguanine is a common premutational lesion formed at non-lex binding sites. These results suggest that CENU derivatives with virtually absolute specificity for A residues would make targeting of lethal, nonmutagenic lesions at A+T-rich regions possible, and this may represent a new strategy for the development of new chemotherapeutic agents with a higher therapeutic index.

Characterisation of circulating chromogranin A in human cancer patients.

The structure of circulating chromogranin A (CgA) of phaeochromocytoma patients was characterised and compared with that of CgA extracted from tumours. Size exclusion chromatography experiments provided evidence that CgA is present in the blood of different patients, as well as in tumour extracts, as multiple forms having different hydrodynamic sizes of 600 kDa (CgA-I), 100 kDa (CgA-II) and 55 kDA (CgA-III). The amount of each CgA form as a proportion of the total antigenic material was different in different patients. Western blot analysis of chromatographic fractions indicated that these forms are made up by polypeptides of similar molecular weight (about 60-70 kDa). All CgA forms express the epitopes recognised by two monoclonal antibodies (A11 and B4E11), directed against residues 68-70 and 81-90 of human CgA. However, their relative immunoreactivity was markedly different. No evidence for the presence of multimeric complexes in the CgA-I fraction was obtained by various immunological and biochemical methods. These results suggest that circulating CgA in phaeochromocytoma patients consists of at least three forms that appear to be made up by polypeptides with similar molecular weight and different hydrodynamic properties and immunoreactivity. We hypothesise that different conformations and shapes contribute to the heterogeneity of circulating CgA.

Reaction of N-(2-chloroethyl)-N-nitrosoureas with DNA: effect of buffers on DNA adduction, cross-linking, and cytotoxicity.

N-(2-Chloroethyl)nitrosoureas (CNU) are clinically used anticancer drugs whose cytotoxicity is associated with the generation of DNA interstrand cross-links. While studying the sequence selectivity for a series of CNU, a dramatic increase in the formation of N7-alkyldeoxyguanosine was observed when Tris buffer was used rather than phosphate or cacodylate buffers. Moreover, the formation of N7-alkyldeoxyguanosine lesions continues in Tris long after all of the CNU has hydrolyzed. These effects are not seen with the monofunctional alkylating analogues, e.g., N-methyl- and N-(2-hydroxyethyl)-N-nitrosourea. In order to determine if the nature of the CNU-mediated DNA damage was altered by Tris, studies were initiated on the following: (1) alkylation of N7-G in end-labeled DNA restriction fragments; (2) covalent modification of DNA with [ethyl-3H]-N-(2-chloroethyl)-N-nitrosourea; and (3) cytotoxicity in L1210 cells. The data presented demonstrate that Tris increases the yield of the "normal" CNU monofunctional cross-linked adducts, i.e., N7-(2-hydroxyethyl)deoxyguanosine, N7-(2-chloroethyl)deoxyguanosine, O6-(2-chloroethyl)deoxyguanosine, and bifunctional adducts, i.e., 1-(deoxycytid-3-yl)-2-(deoxyguanosin-1-yl)ethane and 1,2-bis(deoxyguanosin-7-yl)ethane. In addition, CNU appears to react with Tris to give a long-lived alkylating intermediate that affords large amounts of DNA adducts not seen with CNU in the absence of Tris. However, in vivo toxicity of CNU in L1210 cells is not affected by the presence of Tris, indicating that the reaction pathway(s) responsible for cross-linking is not significantly sensitive to the nature of the buffer.

DNA crosslinking, sister chromatid exchange and cytotoxicity of N-2-chloroethylnitrosoureas tethered to minor groove binding peptides.

Chloroethylnitrosoureas (CENU) are clinically important chemotherapeutic agents whose mechanism of action involves the formation of interstrand DNA crosslinks via an ethane bridge between N1-G and N3-C. CENU generally alkylate G at the N7- and O6-positions, with the latter lesion being the precursor to the interstrand crosslink. In previous studies, we reported the synthesis of CENU appended by a C2H4 linker to the N-terminus of DNA minor groove binding dipeptides (lex, information reading peptides) based on N-methylpyrrole-carboxamide subunits. Because of the dipeptide structure, these CENU-lex's react with DNA at adenines associated with lex equilibrium binding sites. No other CENU has been reported to yield A adducts. The biological evaluation of these CENU-lex's show that they are somewhat less cytotoxic than their simpler counterparts. In addition, in vitro studies show that the minor groove binding CENU-lex's afford a lower level of sister chromatid exchange (SCE) in 9L cells that are sensitive to CENU. There is no difference between CENU-lex in SCE induction in 9L-2 cells that are resistant to CENU. Formation of DNA interstrand crosslinks from the CENU-lex's is lower than for their nonaffinity binding analogs in low ionic strength buffer, but similar in the same buffer containing 200 mM NaCl. Salt inhibits crosslinking for all CENU, but distamycin, a competitive inhibitor of lex minor groove binding, uniquely enhances crosslinks for the CENU-lex's. These results are consistent with the novel minor groove adduction being a 'detoxification' pathway for the CENU-lex's since this lesion is formed at the expense of the cytotoxic major groove interstrand crosslink.

Control over the sequence specificity of DNA alkylation: syntheses and reactions with 32P-end-labelled DNA of N-alkyl-N-nitrosoureas linked to minor groove binding lexitropsins.

The syntheses of N-2-chloroethyl-N-nitrosoureas (Cl-ENU) that are covalently linked to a series of minor groove binding lexitropsins related to distamycin A are reported. The lexitropsins of 2-Cl-ENU show a sequence specificity for alkylating an adenine toward the ends of its DNA affinity binding domains. The reaction of DNA with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea does not yield these products. Therefore, the linking of the 2-Cl-ENU to the minor groove binder qualitatively and quantitatively alters the DNA observed.