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Journal URL: http://www.spandidos-publications.com/or/ We have previously reported the specific binding of nuclear factor AP-1 isolated from human breast MDA MB 468 and HeLa cervical tumor cell lines to oligonucleotides complementary to three newly elucidated sequences within the HIV-1 LTR. These synthesized oligonucleotides, which bear high homology to the AP-1 recognition sequence, were used in the present study in gel retardation assays together with unfractionated nuclear protein extracts from human lung, bladder and head and neck tumors and adjacent normal tissue to study the role of the AP-1 protein in the regulation of HIV-1 expression. We found increased binding of AP-1 to these oligonucleotides in 9/12 lung tumors, 9/14 bladder tumors and 7/7 head and neck tumors as compared to adjacent normal tissues. This confirms previous results obtained when using MDA MB 468 and HeLa nuclear protein extracts. These results indicate that, AP-1 could be contributing to the HIV-1 transcriptional regulation through its interaction with the AP-1 binding sites of HIV-1 LTR.

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Journal URL: http://www.spandidos-publications.com/or/ Infections caused by Human Cytomegalovirus (HCMV) are very common in patients who undergo immunosuppression or immunocompromisation. The techniques used for routine HCMV detection are timeconsuming and lack specificity and sensitivity. The ability of the Polymerase Chain Reaction (PCR) to amplify HCMV DNA from clinical samples of the patients is a valuable diagnostic tool for the detection of HCMV in the early stages of the infection. We used a pair of primers to amplify a 435 bp region of the immediate early-1 gene, to detect HCMV DNA in clinical samples from patients at high risk for HCMV infection. We found HCMV in the following type of patients: 6 out of 20 in immunosuppressed, 11 out of 31 in immunocompromised, 5 out of 8 in pregnant women, 4 out of 25 in patients with high anti-CMV IgM and IgG titres, 1 out of 2 in patients with kidney failure, and 6 out of 14 in patients with opthalmic disorders. Sixty-seven specimens, which were found to be negative for CMV by the PCR technique, were used to inoculate human fibroblast monolayer cultures and PCR was performed to the DNA extracted from the cultured cells. Only in 1 out of the 67 cases HCMV DNA was detected.

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Journal URL: http://www.spandidos-publications.com/or/ Investigation of the nucleotide sequence of the HIV-1 LTR showed the presence of four novel short DNA regions which are homologous to the recognition site for the cellular transcription factor AP-1. Four short oligonucleotide hybrids containing these potential AP-1 sites were constructed and used in gel retardation assays and in competition experiments in order to determine the role of the AP-1 protein in the regulation of HIV-1 expression. The breast MDA MB 468 and cervical HeLa tumor cell lines, which are known to overexpress the AP-1 protein were used in a gel retardation assay as a control to study the affinity of the AP-1 to synthesized oligonucleotide sequences. We have observed specific binding of nuclear factor AP-1 to three of these oligonucleotide hybrids. These results demonstrate the presence of three novel AP-1 binding sites on HIV-1 LTR, one of which was found within the TAR element and in the Tat protein binding region. Moreover, they suggest that AP-1 could be contributing to HIV-1 transcriptional regulation through its interaction with the AP-1 binding sites of HIV-1 LTR.

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Journal URL: http://www.spandidos-publications.com/ijo/ There is evidence that hormone regulation of cellular oncogenes plays an essential role in human cancer. The c-H-ras gene is implicated through both mutation and abnormal gene expression in many types of human cancer. Computer scanning of this gene has revealed two putative hormone response motifs: A possible Glucocorticoid Response Element (GRE) at position 1261 of the first intron of the H-rasl gene and a putative Estrogen Response Element (ERE), at position 3007 of the fourth intron of the gene. In DNA binding assays, using the HeLa and LATK- cell lines, we showed specific binding of the corresponding receptors at both putative Η-ras glucocorticoid and estrogen response sequences, suggesting that hormones could be contributing to Η-ras transcriptional regulation through interaction with their corresponding Hormone Response Elements (HREs).

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Journal URL: http://www.spandidos-publications.com/ijo/ In the context of the hypothesis that the mitochondria represent primary sites of steroid hormone action we have evaluated the possible role of two nucleotide sequences partially homologous to Glucocorticoid Responsive Elements (GREs) present in the cytochrome oxidase subunit I gene of mouse and human mitochondrial DNA, as binding sites for mitochondrial and nuclear regulatory protein(s). Using a gel retardation assay, we examined whether proteins contained in mitochondrial and nuclear extracts from untreated and dexamethasone treated MCF7 and LATK' cell lines bind to these oligonucleotides. Both the mitochondrial and nuclear extracts from these cell lines contain protein(s) that bind to the oligonucleotides, as well as to the GRE of the human metallothionein IIA promoter, binding which was considerably enhanced when the extracts were derived from cells previously treated with dexamethasone.

Journal URL: http://www.spandidos-publications.com/or/ Chronic obstructive pulmonary disease (COPD) is a relatively common disease, affecting mainly males in the western world. Although substantial data are available as regards the clinicopathological characterization of COPD, little is known of the molecular basis of the disease. In the present study we analysed the incidence of microsatellite instability (MI) in cytological specimens from patients with COPD. MI reflects increased mutational rate and is associated with decreased accuracy in the DNA repair, resulting in the accumulation of somatic mutations in cells manifesting this genetic alteration. Among 31 specimens tested, 7 (23%) exhibited MI in at least one among 6 microsatellite markers tested. 5 cases were affected in only one marker while the remaining two cases exhibited evidence of MI in two microsatellite markers. These data suggest that an elevated mutational rate as reflected by the increased incidence of MI is associated with the development of the disease.

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Journal URL: http://www.spandidos-publications.com/or/ DNA extracted from the blood of immunosuppressed and immunocompromised individuals and from patients with infectious mononucleosis, leukaemias and lymphomas were studied using the Polymerase Chain Reaction (PCR) technique. The oligonucleotide primers used for the detection of the Epstein-Barr virus (EBV) amplify a 375bp sequence from the EcoRI Β fragment of the viral genome. EBV specific sequences were amplified from the blood samples of 18 out of 65 patients, most of which were transplant patients (9 out of 31 ). The results confirmed the association of EBV with clinical disorders in immunodeficient and immunocompromised patients and the importance of PCR method in routine diagnosis.

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Journal URL: http://www.spandidos-publications.com/ijo/ ras genes are thought to play an important role in human cancer since they have been found to be activated frequently in several types of human tumors. From preliminary studies it has been found however, that ras mutations are extremely rare in breast tumors and therefore it was of interest to examine the frequency of such mutations. In this study we examined 65 cases of primary breast carcinomas from paraffin blocks, for the presence of point mutations in codons 12 of the K-ras and Η-ras genes. The polymerase chain reaction (PCR) technique was used to amplify a codon 12 containing 157 bp and a 312 bp region of the K-ras and the Η-ras genes respectively, followed by restriction fragment length polymorphism (RFLP) analysis to identify the point mutations. Eight out of the 65 tumors (12.3%) were found to carry a K-ras mutation in codon 12 but none was found to carry a Η-ras mutation. It is suggested that the mutational activation of the K-ras gene may be involved in the development of a small percentage of breast tumors.

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Journal URL: http://www.spandidos-publications.com/ijo/ Ras family genes (H-, K- and N-ras) are implicated in a wide range of human tumours. Mutations are a major activating mechanism for the ras family genes, mainly in codons 12, 13 and 61, resulting in their conversion from proto-oncogenes to activated oncogenes. The detection of mutant ras alleles in human tumours has been performed by several investigators in a wide range of tissues. The aim of our review was to summarize the data obtained from these studies and to investigate whether the presence of mutant ras alleles was associated with particular clinical parameters.

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Journal URL: http://www3.interscience.wiley.com/journal/117983344/

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Journal URL: http://www.spandidos-publications.com/ijo/ The binding activity of the AP-1 FOS/JUN complex to a negative regulatory element on the c-myc promoter was investigated and correlated with events characteristic of terminal differentiation of HMBA-induced mouse erythroleukemic cells. It is suggested that the altered AP-1 activity that was found upon induction of the erythroid differentiation may contribute to the late decline in c-myc mRNA.

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Journal URL: http://www.spandidos-publications.com/ijo/ Bladder cancers are usually curable, by surgical or transurethral excision, if diagnosed at an early stage. Tumor derived mutations in oncogenes potentially provide specific markers for the detection of surgically resectable tumors. The detection of point mutations of Η-ras oncogene correlated with this disease. DNA sequences produced by the Polymerase Chain Reaction (PCR) can be considered for this application, because theoretically bladder tumors should shed cells containing this mutation into the urine. We examined urine from 21 individuals with bladder cancer before any treatment, as well as tissue specimens from the excised tumor and we found 10 mutations of the Η-ras gene at codon 12 in the urine (47.61%) and 14 mutations in the tumor specimens (66.66%). We were able to detect nearly 50% of the patients with bladder tumors using this method. We also studied two relapses; in one case (which presented the mutation in the original tumor and the urine) the relapse grade had progressed from II to III. In the other case the relapse grade stayed at III but it presented for the first time the studied mutation in the urine. These results provide the theoretical and technical basis for the detection of bladder tumors by a non-invasive method and possibly for the evaluation of the invasiveness of the disease.

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Journal URL: http://www.spandidos-publications.com/or/ Complete inactivation of the human retinoblastoma gene is believed to be an essential step in tumorigenesis of several different cancers. Using the plasmid pRbCAT2 that contains the Rb promoter region was tested for its ability to promote transcription of the bacterial chloramphenicol acetyltransferase (CAT) gene in a transient expression assay. This plasmid was co-transfected in a short term transfections with the plasmids pH06Tl and pHO6Nl that contains the mutant and normal Η-ras gene respectively, into the human cell line HeLa, by the calcium phosphate technique. It was found that the mutant Η-ras gene enhances the activity of the Rb gene promoter in contrast to the normal Η-ras gene that inhibits it. The expression of the CAT gene in stable clones of HeLa cells carrying the promoter of Rb gene after treatment with TPA and EGF respectively, was also investigated, whereas TPA enhanced, EGF had no effect on the activity of the Rb gene promoter.

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