A. Zafiropoulos

Significant involvement of CCR2-64I and CXCL12-3a in the development of sporadic breast cancer.

The molecular biology of cancer is still far from being understood, with the exception of specific familial cases. Amplifications of oncogenes and alterations in tumour suppressor and detoxification genes by mutations or deletions appear especially important in the development of sporadic breast tumours.1–3 Tumour infiltrating lymphocytes (TILs) and tumour associated macrophages are thought to play a crucial role in tumour immune surveillance and possibly development. The activation and recruitment of lymphocytes is regulated by chemotactic and proinflammatory chemokines such as RANTES (CCL5), MCP-1, and to a lesser extent MIP-1 alpha, MIP-1 beta, and IL-8.4–7 It has been suggested that melanoma cells evade immune surveillance through the induction of TIL cell death by SDF-1 alpha (CXCL12) and RANTES.8 MCP-1 is the natural ligand of the CCR2 chemokine receptor, expressed mainly in the monocytes, activated T lymphocytes, and memory cells,9 whereas RANTES is the ligand of CCR5.10 The relevance of chemokines to malignancy extends beyond leucocyte recruitment. Animal models have shown that chemokine secretion by tumour cells can influence angiogenesis and tumour growth. Expression of angiogenic CXC chemokines by tumour cells in severe combined immunodeficient (SCID) syngenic mice has been shown to enhance tumour growth.11 However, the association of any kind of cancer with chemokine related genetic markers has not been examined to date. We selected the polymorphisms CCR2–64I,12 CXCL12–3′A, CCR5Δ32, and CCR5 59029 G-A10,13 because of functional and clinical data from AIDS studies. We determined the genotype for the above four polymorphisms in 442 cancer samples and 361 control samples. Our data indicate a significant involvement of the chemokine system in the development of breast cancer. ### Patients and samples #### Breast cancer Blood samples were collected at the Prolepsis (centre for breast cancer diagnosis and research in Athens, Greece) from 233 female patients with breast cancer, …

Transforming Growth Factor‐β as a key molecule triggering the expression of versican isoforms v0 and v1, Hyaluronan Synthase‐2 and synthesis of Hyaluronan in Malignant Osteosarcoma cells

Versican, a large sized chondroitin‐sulphate proteoglycan (PG), and its binding partner, hyaluronan (HA), are extracellular matrix (ECM) components that play an essential role in transformed cell behavior. Expression of certain versican isoforms has been implicated in cell migration and proliferation of cancer cells and, on the other hand, disruption of HA synthesis by inhibiting hyaluronan synthase‐2 (HAS2) expression in osteosarcoma cells by suppressing cell proliferation, invasiveness and motility. Considering that growth factors, such as TGF‐β, bFGF and PDGF‐BB, are important regulators for the expression of the ECM macromolecules, in this study we examined the effect of these growth factors on the expression of the various versican isoforms, HA synthases as well as HA synthesis by MG‐63 osteosarcoma cells and normal human osteoblastic periodontal ligament cells (hPDL). Real‐time PCR and metabolic labelling followed by fine HPLC analysis coupled to radiochemical detection were the methods utilized. It was found that, contrary to normal hPDL cells, osteosarcoma MG‐63 cells do not constitutively express the versican isoforms V0 and V1. Exogenous addition of TGF‐β2 stimulated the versican transcript levels mainly by forcing osteosarcoma cells to express V1 and V0 isoforms. PDGF‐BB and bFGF had only minor effects in these cells. In hPDL cells a strong stimulation of the V3 transcript by all growth factors was observed. TGF‐β2 was also the major stimulator of HAS2 isoform expression as well as hyaluronan synthesis in osteosarcoma cells, while PDGF‐BB exerted dominant influence on HAS2 isoform expression and hyaluronan biosynthesis by osteoblasts. The obtained results show for the first time that TGF‐β2 triggers the malignant phenotype pattern of versican and hyaluronan expression in human osteosarcoma cells and indicate that this growth factor may account for the metastatic potential of these cells. IUBMB Life, 58: 47 ‐ 53, 2006

Mutational analysis of CDKN2A genes in patients with squamous cell carcinoma of the skin

Summary Background Nonmelanoma skin cancers [squamous cell carcinomas (SCC) and basal cell carcinomas (BCC)] are the most common neoplasias of the Caucasian population.

Relation of PON1 and CYP1A1 genetic polymorphisms to clinical findings in a cross-sectional study of a Greek rural population professionally exposed to pesticides

Allelic variants of CYP1A1 and PON1 have been extensively studied as susceptibility factors in toxic response, although little is known about the role of these variants as risk factors for the plethora of diseases appearing in the human population. In this study we investigated the hypothesis of correlation of CYP1A1 and PON1 enzymes with the incidence of various medical examination findings in a Greek rural population professionally exposed to a variety of pesticides. The medical history of 492 individuals, randomly selected for the total population of 42,000, was acquired by interviews and their genotype determined for the CYP1A1*2A, PON1 M/L and PON1 Q/R polymorphisms. The assessment of exposure to pesticides of the population was verified by analytical methods. Analysis of the genetic data revealed that the allele frequencies of PON1 R, M and CYP1A1*2A alleles were 0.243, 0.39 and 0.107 respectively. The CYP1A1*2A polymorphism was found to have significant association with chronic obstructive pneumonopathy (p=0.045), peripheral circulatory problems (trend p=0.042), arteritis (p=0.022), allergies (trend p=0.046), hemorrhoids (trend p=0.026), allergic dermatitis (p=0.0016) and miscarriages (p=0.012). The PON1 Q/R polymorphism was found to have significant association with hypertension (p=0.046) and chronic constipation (p=0.028) whereas, the L/M polymorphism, with diabetes (p=0.036), arteritis (trend p=0.022) and hemorrhoids (trend p=0.027). Our results demonstrated an association between the CYP1A1/PON1 polymorphisms and several medical examination findings, thus indicating the possible involvement of the human detoxification system to health effects in a rural population exposed professionally to pesticides.

Human herpes viruses in non-melanoma skin cancers

We examined the possible involvement of human herpes viruses in sporadic non-melanoma skin cancer of Greek patients. Polymerase chain reaction (PCR) based detection assays were utilized for the detection of viral cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) genomes in 24 squamous cell carcinomas (SCC), five Bowens disease, 72 basal cell carcinomas (BCC) specimens and eight premalignant lesions. Forty-two of 109 (38.5%) skin lesions were found positive for CMV DNA. The highest incidence was 6/8 (75%) observed in specimens with premalignant lesions. The incidence was 37.5% (27/72) in BCC, 33% (8/24) in SCC and 20% (1/5) in extragenital Bowens disease. All samples were negative for HSV-1/2 and EBV DNA as assessed by our PCR based assay. The CMV infection showed no statistically significant correlation with the histological type, age, site of lesion or sex. Our results give a strong indication of the possible involvement of CMV in non-melanoma skin cancer development.

Association between schizophrenia and DRD3 or HTR2 receptor gene variants.

Schizophrenia is a severe and common psychiatric disorder afflicting 1% of the world population. A role of many neurotransmitter receptors in schizophrenia was suggested by an association with several polymorphisms located in their coding regions. In this study we examined the contribution of the T-102C and A-206G transitions in the 5-HTR2a and DRD3 receptor genes respectively to genetic susceptibility and phenotypic expression of schizophrenia disorder within the Greek population. We determined by PCR and RFLP analysis the genotype for the above polymorphisms in 114 schizophrenic hospitalized individuals and 192 control samples. In contrast to previous reports from large European multicentre studies, which indicate significant correlation between schizophrenia and C-102 allele of the T-102C polymorphism, in this study we observed a statistically significant overall association between the disorder and allele T-102 (P<0.0001, odds ratio (OR)=2.11, 95% CI=1.48–3.02). We also found a highly significant excess of the T-102/C-102 and C-102/C-102 genotypes in the normal group (P<0.001). Comparison of the patients with the controls for the DRD3 polymorphism (A-206G transition) showed marginally nonsignificant differences in the genotypic (P=0.054) and no significance in the allelic (P=0.163) frequencies. However, the A-206/A-206 genotype seems to positively contribute to the disorder appearance, when compared to A-206/G-206 as genotype base line risk (P=0.016, OR=1.88, 95% CI=1.09–3.26). In conclusion, from genetic association analysis of this schizophrenic population, a significant association is clearly determined between the HTR2 genetic polymorphism and the presence of schizophrenic disorder, manifested as increased risk of schizophrenia for carriers of the T-102 allele.

Ionic interaction of the HIV-1 V3 domain with CCR5 and deregulation of T lymphocyte function

We have reported that the principal neutralizing domain of V3 of the HIV-1 gp120 induces an antigen-specific activation apoptosis of responding effector CD4+ T lymphocytes, a phenomenon inhibited by RANTES, an agonist of CCR5. Here, addressing the question of how a hypervariable region could induce such a selective reaction, we demonstrated that the magnitude of the activation phase was dependent on the number of basic amino acids present in the V3 peptide, an observation confirmed by using V3 peptides with appropriate basic amino acid substitutions. The relative position of the amino acids in the V3 peptide did not affect the biological phenomenon. Using surface plasmon resonance biosensor analysis, we also provided direct evidence of the influence of basic amino acids in the interaction between V3 and the amino terminal domain of CCR5. Sulphation of tyrosines in the CCR5 peptide was essential. Our results confirm gp120 modelling predictions and demonstrate simple molecular ionic interactions as capable of affecting key cell events, the wider biological implications of which need to be further explored.

Cardiotoxicity in rabbits after a low-level exposure to diazinon, propoxur, and chlorpyrifos:

Lethal cardiac complications leading to death and various arrhythmias have been reported after organophosphate and/or carbamate poisonings. The present study focuses on the long-term effects of repeated low-level exposure to diazinon, propoxur, and chlorpyrifos (CPF) on cardiac function in rabbits. The yearly based experimental scheme of exposure consisted of two oral administration periods, lasting 3 months and 1 month each, interrupted by an 8-month washout period (total duration 12 months). At the end of the experimental scheme, the rabbits underwent an echocardiographic evaluation under sedation, after which they were killed and the tissue and serum samples were collected. A mild localized cardiotoxic effect was established by echocardiography for the three pesticides tested. Severe histological alterations were identified, especially in the diazinon-treated animals in agreement with increased persistence of this pesticide established in the cardiac tissue. In addition, all pesticides tested increased the oxidative stress and oxidative modifications in the genomic DNA content of the cardiac tissues, each one following a distinct mechanism.

Chondroitin sulfate prevents platelet derived growth factor-mediated phosphorylation of PDGF-Rβ in normal human fibroblasts severely impairing mitogenic responses

Platelet‐derived growth factor (PDGF) is a major polypeptide mitogen for cells of mesenchymal origin such as fibroblasts. Chondroitin sulfate chains (CS), which are abundant in the extracellular matrix have been shown to physically interact with PDGF‐BB modulating its biological function. The aim of the present study was to examine the involvement of CS on PDGF‐BB induced proliferative responses and receptor activation in human lung fibroblasts. The addition of exogenous free CS chains caused a significant downregulation of the PDGF‐BB mediated mitogenic and chemotactic responses. Similar results were obtained by the increase of endogenous CS biosynthesis after β‐D‐xyloside treatment. Furthermore, removal of the membrane‐bound CS chains by selective enzymatic treatment significantly increased the proliferative capacity of human fibroblasts. Analysis of PDGF‐R phosphorylation in the presence of CS or β‐D‐xyloside, revealed a reduction of PDGF‐Rβ phosphorylation in the tyrosine residue 1021. These results demonstrate, for the first time, that CS either soluble or surface bound downregulates the mitogenic responses of PDGF‐BB in normal human lung fibroblasts through the reduction of PDGF‐Rβ phosphorylation. J. Cell. Biochem. 103: 1866–1876, 2007.

Detection of point mutations at codon 12 of KI-ras in ophthalmic pterygia

AbstractAimsOphthalmic pterygium is a potentially vision-threatening lesion of unknown etiology, related to an exposure to solar light. Mutations to the ras genes are frequently observed in lesions related to an exposure to solar light. The present study aims at screening pterygia for mutations at codons 12 and 13 of the ras genes.MethodsIn all, 50 pterygia were examined, together with respective blood samples and specimens of normal conjunctiva. A PCR reaction was performed to amplify sequences containing codons 12 and 13 of Ki-ras, H-ras, and N-ras. An RFLP analysis was then performed to detect point mutations at codon 12. The mutational status at codons 12 and 13 was further explored with sequencing of PCR products.ResultsRFLP analysis revealed Ki-ras mutations at codon 12 in five (10%) of pterygia, whereas H-ras or N-ras mutations were not observed. Sequencing confirmed Ki-ras mutations at codon 12 and revealed absence of mutations at codon 13. The presence of Ki-ras mutations was significantly correlated with postoperative recurrence (P=0.02) and young age (P=0.04). Mutations were not observed in specimens of blood or normal conjunctiva for any of the genes examined.ConclusionsThe absence of N-ras mutations is in agreement with previous reports concerning mucosal lesions. The detection of Ki-ras mutations and the association with postoperative recurrence implies a possible role of Ki-ras in the clinical profile of pterygium. The mechanism of Ki-ras mutations is unclear and could be independent of the action of UV light.