Evaluation of O-alkyl and aryl sulfonyl aromatic and heteroaromatic amidoximes as novel potent DNA photo-cleavers.

Several stable O-alkyl and aryl sulfonyl conjugated p-nitro-Ph and o-, m-, p-pyridine N'-hydroxy imidamides, were subjected to UV irradiation at 312 nm with supercoiled circular plasmid DNA pBluescript KS II. The generated amidinyl and sulfonyloxyl radicals led to effective DNA photo-cleavage. Both alkyl and aryl sulfonyl derivatives were active and the order p-pyridine > p-nitro-Ph > o-pyridine > m-pyridine was schematized for the N'-hydroxy imidamides moiety. Calf thymus-DNA affinity studies which comprised UV interactions, viscosity experiments and competitive studies with ethidium bromide showed good to excellent affinity of the compounds. These properties revealed sulfonyl amidoximes as novel effective DNA-photo-cleavers and may serve in the discovery of new leads for "on demand" biotechnological and medical applications.

Synthesis of folate- pegylated polyester nanoparticles encapsulating ixabepilone for targeting folate receptor overexpressing breast cancer cells.

The aim of this study was the preparation of novel polyester nanoparticles based on folic acid (FA)-functionalized poly(ethylene glycol)-poly(propylene succinate) (PEG-PPSu) copolymer and loaded with the new anticancer drug ixabepilone (IXA). These nanoparticles may serve as a more selective (targeted) treatment of breast cancer tumors overexpressing the folate receptor. The synthesized materials were characterized by (1)H-NMR, FTIR, XRD and DSC. The nanoparticles were prepared by a double emulsification and solvent evaporation method and characterized with regard to their morphology by scanning electron microscopy, drug loading with HPLC-UV and size by dynamic light scattering. An average size of 195 nm and satisfactory drug loading efficiency (3.5%) were observed. XRD data indicated that IXA was incorporated into nanoparticles in amorphous form. The nanoparticles exhibited sustained drug release properties in vitro. Based on in vitro cytotoxicity studies, the blank FA-PEG-PPSu nanoparticles were found to be non-toxic to the cells. Fluorescent nanoparticles were prepared by conjugating Rhodanine B to PEG-PPSu, and live cell, fluorescence, confocal microscopy was applied in order to demonstrate the ability of FA-PEG-PPSu nanoparticles to enter into human breast cancer cells expressing the folate receptor.

Distinct distribution of corticotropin releasing factor receptors in human breast cancer.

The hypothalamic neuropeptide corticotropin releasing factor (CRF) has been found in several types of human cancer, where its biological role is not clarified. In experimental models of breast cancer CRF has been shown to exert anti-proliferative and other actions. Aim of the present study was to describe the expression of the two types of CRF receptors CRF(1) and CRF(2) in human breast tumors. Receptor expression was studied in breast biopsies from patients diagnosed for primary breast adenocarcinoma, obtained from the tumor and the adjacent benign tissue. Gene expression levels were evaluated by real-time PCR following reverse transcription of total RNA extracts. CRF(1) transcripts were found in 23.1% of benign and in 23.1% of malignant biopsies. CRF(2(a)) was found in 22.2% of benign and 36.0% of malignant biopsies. Transcript levels of both receptors did not differ significantly between cancer and benign biopsies from the same tumor. No correlation was found between CRF receptor expression and patient histo/clinicopathological characteristics. Histological mapping using immunohistochemistry revealed positive CRF(1) immunostaining in the cancerous implants and breast ducts, whereas CRF(2) immunoreactivity was localized mainly in the perineural invasions. In conclusion, both CRF receptors were found in breast cancer and the respective benign adjacent tissue. The two CRF receptor proteins presented distinct distribution and subcellular localization, pointing into differing biological roles. CRF receptors could serve as targets of endogenous ligands expressed in the tumor microenvironment, regulating cancer growth.

Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway.

The role of herpes simplex virus ICP27 protein in mRNA export is investigated by microinjection into Xenopus laevis oocytes. ICP27 dramatically stimulates the export of intronless viral mRNAs, but has no effect on the export of cellular mRNAs, U snRNAs or tRNA. Use of inhibitors shows, in contrast to previous suggestions, that ICP27 neither shuttles nor exports viral mRNA via the CRM1 pathway. Instead, ICP27-mediated viral RNA export requires REF and TAP/NXF1, factors involved in cellular mRNA export. ICP27 binds directly to REF and complexes containing ICP27, REF and TAP are found in vitro and in virally infected cells. A mutant ICP27 that does not interact with REF is inactive in viral mRNA export. We propose that ICP27 associates with viral mRNAs and recruits TAP/NXF1 via its interaction with REF proteins, allowing the otherwise inefficiently exported viral mRNAs to access the TAP-mediated export pathway. This represents a novel mechanism for export of viral mRNAs.

Herpes simplex virus type 1 blocks the apoptotic host cell defense mechanisms that target Bcl-2 and manipulates activation of p38 mitogen-activated protein kinase to improve viral replication.

Wild-type (wt) herpes simplex virus type 1 (HSV-1) suppresses cell death. We investigated the apoptotic pathways triggered during infection with mutant viruses tsk and 27lacZ (which lack functional ICP4 and ICP27 viral proteins, respectively) and examined the mechanisms used by wt HSV-1 to protect against programmed cell death induced by the DNA-damaging compound cisplatin. In our studies, we used BHK and HeLa cells, with similar results. We suggest that a decrease in the levels of Bcl-2 protein is a key event during apoptosis induced by the mutant viruses and that Bcl-2 levels are targeted by (i) a decrease of bcl-2 RNA, (ii) caspase-related proteolysis, and (iii) p38 mitogen-activated protein kinase (p38MAPK)-dependent destabilization of Bcl-2 protein. We show that wt HSV-1, but not the mutant viruses, maintains bcl-2 RNA and protein levels during infection and protects from the cisplatin-induced decrease in bcl-2 RNA; our data suggest that both ICP27 and ICP4 are required for this function. Additionally, wt HSV-1 evades but does not actively block activation of caspases. Although wt HSV-1 induces p38MAPK activation during infection, it prevents p38MAPK-dependent destabilization of Bcl-2 and exploits p38MAPK stimulation to enhance transcription of specific viral gene promoters to increase viral yields.

The human papillomavirus type 16 negative regulatory RNA element interacts with three proteins that act at different posttranscriptional levels.

In human papillomaviruses, expression of the late genes L1 and L2, encoding the capsid proteins, is restricted to the upper layers of the infected epithelium. A 79-nt GU-rich negative regulatory element (NRE) located at the 3' untranslated region of the human papillomavirus 16 L1 gene was identified previously as key to the posttranscriptional control of late gene expression. Here, we demonstrate that in epithelial cells, the NRE can directly bind the U2 auxiliary splicing factor 65-kDa subunit, the cleavage stimulation factor 64-kDa subunit, and the Elav-like HuR protein. On induction of epithelial cell differentiation, levels of the U2 auxiliary splicing factor 65-kDa subunit decrease, levels of the cleavage stimulation factor 64-kDa subunit increase, and the levels of HuR remain unchanged, although redistribution of the HuR from the nucleus to the cytoplasm is observed. Late gene transcripts, which appear to be fully processed, are detected in undifferentiated W12 cells, but are confined in the nucleus. We propose that repression of late gene expression in basal epithelial cells may be caused by nuclear retention or cytoplasmic instability of NRE-containing late gene transcripts.

Detection of herpes simplex virus (HSV) in aborted material using the polymerase chain reaction technique.

OBJECTIVE: To investigate the contribution of HSV to the aetiopathogenesis of spontaneous abortion. DESIGN: A hospital-based, case-control study. SETTING: Department of Obstetrics and Gynecology, University Hospital and Laboratory of Clinical Virology, Medical School, University of Crete, Heraklion, Crete, Greece. POPULATION AND METHODS: Abortion material from 102 cases of women with spontaneous abortion was analysed for the presence of HSV DNA applying the PCR technique. Serological assays were used for the detection of specific IgM and IgG antibodies in the maternal sera of 90 pregnant women with successful outcome of their pregnancy while 70 non-pregnant women at reproductive age were also examined as control. RESULTS: The HSV genome was detected by PCR amplification in 3 cases of spontaneous abortion, 2 of them exhibited serological markers of virus reactivation while the 3rd showed a past infection. There were no obvious clinical manifestations indicating a current herpes infection. Both groups of pregnant women, either with spontaneous abortion or with a successful outcome of pregnancy, displayed serological markers of HSV reactivation at higher rates compared with non-pregnant women (chi2, p < 0.05). CONCLUSIONS: Using the PCR technique we were able to detect the HSV genome in gestational tissues of spontaneous abortions, even in cases without any clinical symptoms or seropositivity for a primary infection. Serological assays were not very useful for the elucidation of the role of HSV in inducing spontaneous abortions, although they indicate that the state of pregnancy predisposes to HSV reactivation. However, the detection of HSV in 3 out of a total number of 102 cases does not support HSV infection as a major abortion-related factor.

Evaluation of Parvo B19, CMV and HPV viruses in human aborted material using the polymerase chain reaction technique.

OBJECTIVE: To investigate the role of human parvovirus B19 (Parvo B19), cytomegalovirus (CMV) and human papilloma virus (HPV) viruses in the aetiopathogenesis of spontaneous abortions. STUDY DESIGN: Abortion material from 102 cases of women with spontaneous abortions were analysed for the presence of Parvo B19, CMV and HPV DNA using the polymerase chain reaction (PCR) technique. Serological assays were used for the detection of specific IgM and IgG antibodies against Parvo B19 virus and CMV in the maternal sera. RESULTS: Parvo B19 virus genome was detected in two cases of spontaneous abortion, by PCR amplification, while CMV and HPV genomes were not observed. Serological markers were indicative for Parvo B19 virus and CMV infection in ten and four cases, respectively. CONCLUSIONS: PCR is a useful method for investigating the viral contribution to the aetiopathogenesis of spontaneous abortions and for detecting the viral genome in the abortion material. This study of 102 cases of spontaneous abortion does not implicate CMV and HPV in the aetiopathogenesis of spontaneous abortion, although it indicates a possible abortional role for Parvo B19 virus.

Oncogenes and onco-suppressor genes in female genital cancer.

Cancer is a multistep process resulting in the accumulation of genetic lesions in proto-oncogenes or tumor suppressor genes. In recent years, many biological studies have focused on the analysis of the genetic and molecular events occurring in genital tumors in order to identify genes involved in their initiation and progression. Understanding the genetic events that lead to initiation and progression of a disease remains an important challenge in gynecological research and ultimately may enable the development of better approaches for earlier diagnosis, in cases where current therapeutic strategies have a high cure rate.

Cervical cytology in adolescence.

The prevalence of cervical intraepithelial neoplasia in young women is increasing worldwide. Frequency of the abnormal Papanicolau smears was examined in sexually active young females aged 13 to 19 years. The smears were reported as within normal limits or with benign cellular changes due to inflammation in 96.2% of cases. The cytological changes were related in 2.5% of cases of HPV infection, in 1.0% of cases to CIN I and in 0.3% of cases to CIN II. These results show a frequency of 3.8% of abnormal smears.

Codon 12 ras mutations in patients with myelodysplastic syndrome: incidence and prognostic value.

To determine the prevalence of activated rasoncogenes (N-ras, Harvey-ras Kirsten-ras), DNA derived from peripheral blood of 51 patients with myelodysplastic syndrome (MDS) was investigated. The method was based on the polymerase chain reaction (PCR) technique to amplify DNA, followed by restriction fragment length polymorphism (RFLP) analysis. Among the French-American-British (FAB) subtypes, N-ras mutations were found in two patients with refractory anemia with excess of blasts (RAEB), in one patient with refractory anemia with excess of blasts in transformation (RAEB-t), and in two patients with chronic myelomonocytic leukemia (CMML). MDS patients with a mutation at codon 12 of the N-ras gene showed shorter survival duration than other MDS patients of the same FAB subtypes, although these findings proved to be not statistically significant (P > 0.1). Interestingly, all but one patient with N-ras mutation developed acute myelogenous leukemia (AML). In conclusion, the presence of mutation at codon 12 of the N-ras gene might serve as a negative prognostic factor at diagnosis of MDS.

ras gene mutations in human endometrial carcinoma.

The purpose of this study was to assess the extent of involvement of the ras oncogene activation by point mutations in endometrial carcinoma in the Greek population. The PCR technique was employed, followed by RFLP analysis to identify the point mutations in codon 12 of the K-ras, H-ras and N-ras genes. K-ras gene point mutations were detected in 8 of the 55 cases (15%) of primary endometrial carcinoma, H-ras in 4 (7.3%), while no mutations were found for the N-ras gene. No correlation was found between the presence of ras gene mutations and the clinicopathological parameters, or patient survival. The only association found was between H-ras mutations and the FIGO stage of the tumor (Fisher's exact test, p = 0.011). These results indicate a possible role of ras gene activation in a small subset of endometrial carcinomas.

The association of the H-ras oncogene and steroid hormone receptors in gynecological cancer.

Expression of the ras family of cellular oncogenes is associated with tumorigenicity, invasiveness and metastatic potential in a variety of human carcinomas. Additionally, H-ras cooperates with glucocorticoids and with ovarian hormones in cell transformation and in the development of mammary carcinomas. Steroids are considered to be tumor promoters and their levels influence the cure rates and survival of the patients with gynecological lesions. It is proposed that they exert tumor promoting activity by transcriptional regulation of nuclear proto-oncogenes, such as c-fos, c-jun, and c-myc. The human H-ras gene contains within its first and fourth introns, sequences that are specifically recognized by glucocorticoid and estrogen receptors, respectively. Using gel retardation assays, the level of steroid receptor binding in H-ras elements has been compared, employing nuclear extracts from human endometrial and ovarian lesions and from the adjacent normal tissue. Elevated binding of the glucocorticoid and estrogen receptors in the corresponding H-ras elements in almost all tissue pairs tested has been found. It is suggested that the H-ras proto-oncogene is hormonally regulated and directly implicated in human gynecological cancer through elevated, steroid-induced gene expression.

Glucocorticoid and estrogen receptors have elevated activity in human endometrial and ovarian tumors as compared to the adjacent normal tissues and recognize sequence elements of the H-ras proto-oncogene.

We examined the level of receptor binding in H-ras elements, using nuclear extracts derived from human endometrial and ovarian lesions and from adjacent normal tissue in gel retardation assays. We found increased binding of the glucocorticoid receptor (GR) to the H-ras GR element in more than 90% of endometrial tumors and in all ovarian tumors tested, as compared to the corresponding adjacent normal tissue. Additionally, we found elevated binding of the estrogen receptor (ER) in H-ras ER element in all pairs of ovarian tumor/normal tissue tested, whereas in ER-negative control breast tumor/normal tissue pairs, no differences in ER DNA-binding levels were observed. These results suggest that steroid hormone receptor binding could directly activate the H-ras oncogenic potency in human endometrial and ovarian lesions, providing additional evidence for the role of H-ras expression in hormonally responsive human cancers.

Detection of human papilloma virus (HPV) and K-ras mutations in human lung carcinomas.

The purpose of our study was to assess the prevalence and prognostic significance of HPV infection as well as K-ras codon 12 point mutations in lung cancer. Patients diagnosed with lung carcinoma between 1988 and 1992 (N=99) were selected. HPV detection and typing was performed by PCR from paraffin-embedded tissues, while mutations in codon 12 of K-ras gene were detected using the restriction fragment length polymorphism (RFLP) analysis. The prevalence of HPV infection was 15%, while K-ras codon 12 point mutations were found in 18% of the specimens examined. In 50% of the HPV-positive cases, K-ras gene mutation coexisted. HPV 18 was the most frequent type. No correlation was found between K-ras mutation and HPV infection with sex, age and clinical outcome of the patient, or the histological type and the differentiation grade of the tumor. An association was found between K-ms codon 12 point mutations and the stage of the tumor, occurring more frequently at stage III (p=0.037). Infection with potentially oncogenic HPV types could co-operate with K-ras gene activation in the progression of the disease, since K-ras activation by point mutations seems to be a late event in lung carcinogenesis.

Association of herpesvirus infection with the development of genital cancer.

Clinical observations and epidemiological studies on genital cancer have revealed an association with sexual behavior, thus motivating research into sexually transmitted agents which may be responsible for the neoplasia. In this study, we used the PCR technique to examine the presence of CMV, HSV and EBV viruses in 187 cases of human genital lesions and found that infection with CMV or HSV was associated with cervical cancer. When we stratified according to HPV status this association was found only for HPV-DNA-negative cases. These findings indicate that past infection with CMV or HSV could be interpreted as a surrogate marker of HPV infection. However, these viruses may play an important role themselves in cervical cancer.

Hpv detection in stained cytological cervical specimens and correlation with cytology and histology.

It has been suggested that detection of high risk human papillomavirus (HPV) in low grade cervical intraepithelial neoplasia contributes to transition to high grade lesions and cancer. Currently, the PAP smear is the primary screening tool to identify women with cervical disease, specifically cervical intraepithelial neoplasia (GIN). In the present study we examined the utility of HPV detection and typing by PCR, in women with cytological and/or histological evidence of low grade squamous lesion (LGSL), using stained PAP cervical smears. HPV infection was confirmed in 21 out of the 31 (68%) specimens examined.

Detection of ras gene-mutations and hpv in lesions of the human female reproductive-tract.

Compatible with the epidemiology and natural history of cervical cancer and with experimental findings that HPV-immortalized nontumorigenic cells are similar to cervical carcinoma cells, in terms of the quantity and type of viral RNA expressed, it is believed that additional cellular genetic events are necessary for tumorigenic conversion. In the current study we detected codon 12 point mutations of the K-ras oncogene with an incidence of 28.2% in malignant lesions of the cervix, as well as HPV 1 8 at a higher rate than HPV16 (30.2% vs 27.9%) in genital lesions, by PCR and RFLP analysis. Codon 12 point mutations of K-, H- and N-ras were also found in benign lesions of the cervix, in endometrial and in ovarian carcinomas, although at a lower frequency. It is suggested that the mutationally activated ras oncogenes cooperate with HPV in the early stages of carcinogenesis of the human female reproductive tract.

Mutational activation of k-ras oncogene in human breast-tumors.

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 H-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 H-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 H-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.

Detection of human cytomegalovirus by the polymerase chain-reaction in immunosuppressed and immunocompromised patients.

Infections caused by Human Cytomegalovirus (HCMV) are very common in patients who undergo immunosuppression or immunocompromisation. The techniques used for routine HCMV detection are time-consuming 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.