M. Ergazaki

Mutations and Expression of the ras Family Genes in Leukemias

The levels of expression and the incidence of codon 12 point mutation of the ras family genes were studied in 18 cases of leukemia, seven with acute myeloblastic leukemia (AML), three with acute lymphoblastic leukemia (ALL), four cases with chronic myelogenic leukemia (CML) and four cases with chronic lymphocytic leukemia (CLL). Elevated expression of the ras genes was found for 39%, 61% and 67% of the specimens for the H‐ras, K‐ras and N‐ras, respectively. A trend was found between the overexpression of the N‐ras gene and the acute leukemias: all 10 acute leukemias exhibited overexpression of the N‐ras gene, while only two of the CML cases, both in blastic crisis, showed elevated levels of the N‐ras gene. Codon 12 point mutations at the N‐ras gene were found in two of seven cases (28%) with AML and one of the four cases (25%) with CML. The only K‐ras codon 12 point mutation was found in a patient with CLL. No mutations were found in the codon 12 of H‐ras. Our data suggest that apart from the point mutations, overexpression of the ras family genes is important in the development of the disease.

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.

Detection of Epstein-Barr virus and human papillomavirus in nasopharyngeal carcinoma by the polymerase chain reaction technique

We used the PCR technique to detect the Epstein-Barr virus (EBV) and human papillomavirus (HPV) DNA in paraffin-embedded tissues from Greek patients with nasopharyngeal carcinoma (NPC). The oligonucleotide primers used for the detection of EBV amplify a 375-bp long sequence from the EcoRI B fragment of the viral genome, whereas for HPV the primers amplify a 151-bp long sequence of the viral genome. The PCR products were analysed by agarose gel electrophoresis and visualised by UV illumination after staining with ethidium bromide. Sixty-three specimens were examined. EBV specific sequence was amplified in 20 (32%) and HPV in 12 (19%) out of the 63 samples. There was no co-infection with EBV and HPV. Although there is a high correlation of EBV infection with poorly differentiated NPC in patients from Southern China and South-East Asia, the restricted distribution suggests genetic or environmental cofactors in the development of the neoplasm. Our results confirm this suggestion since there was only a 32% correlation of EBV with NPC in Greece. HPV may also be involved in the carcinogenesis of EBV-negative squamous cell nasopharyngeal carcinomas.

Loss of heterozygosity at 9p and 17q in human laryngeal tumours

Recent investigations revealed that the 9p arm and 17q arm of human chromosomes harbour tumour suppressor genes (TSGs) with an important role in multistage carcinogenesis. At the 9p arm is located the p16 (MTS1) TSG and probably others with an effect on various human tumours such as acute lymphoblastic leukaemia, bladder cancer, gliomas, malignant mesotheliomas, melanomas and non-small cell lung carcinomas. In addition, the 17q arm harbours BRCA1 TSG which is responsible for approximately 80% of the familial breast/ovarian cancer cases. In order to investigate the implication of these performed a loss of heterozygosity (LOH) analysis with 10 polymorphic microsatellite markers (three at the 17q arm surrounding the BRCA1 region and seven at the 9p arm). Fourteen of the 17 (82%) tumours exhibited deletions at 9p. The highest incidence of LOH (6/13, 46%) was found for the marker D9S157 at 9p22. One sample exhibited deletion of all the informative markers tested indicating deletion of the complete 9p arm. No homozygous deletions were found. LOH at the 17q arm near the BRCA1 locus was found in 6 (35%) among 17 specimens. The results of this study indicate that allelic deletions at 9p are frequent in the development of laryngeal tumours. The highest incidence of LOH was found for the marker D9S157 which is near, but distinct from the location of p16 (MTS1) tumour suppressor gene, indicating the presence of multiple tumour suppressor genes within this chromosomal region. In addition, BRCA1 TSG is implicated in the development of laryngeal tumours.

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 (χ2, 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.

Detection of Epstein-Barr virus genome in squamous cell carcinomas of the larynx.

Epstein-Barr virus (EBV) is a B-lymphotropic virus with a tumorigenic potential. EBV infection has been recognized as the main cause of nasopharyngeal carcinoma and Burkitts lymphoma. The aim of our study was to determine the incidence of EBV in squamous cell carcinomas of the larynx. We employed for our analysis a sensitive polymerase chain reaction (PCR) assay, followed by restriction fragment length polymorphism (RFLP) for further confirmation of the specificity of the PCR-amplification reaction. Our analysis revealed that 9 of 27 (33%) specimens harbored the EBV genome in the tumor tissue while only 4 (15%) specimens from adjacent normal tissue exhibited evidence of EBV infection. Three were EBV positive for both normal and tumor tissue. No association has been found with disease stage, histological differentiation and nodes at pathology. The relatively high incidence of EBV in the tumor tissue (33%) of patients with laryngeal cancer, as compared to the low (15%) incidence of the virus genome detected in the adjacent normal tissue of the patients, indicates a probable role of EBV in the development of the disease.

Transcriptional activation of the human immunodeficiency virus long terminal repeat sequences by retinoic acid in human epithelial and fibroblast tumor cell lines

We employed a recombinant plasmid, pBHIV1, carrying the long terminal repeat (LTR) sequences of HIV-1 linked to the reporter chloramphenicol acetyl transferase (CAT) gene and to the aminoglycoside phosphotransferase (aph) gene as a selectable marker. We introduced pBHIV1 into human epithelial and fibroblast tumor cell lines (HeLa and MRCSV40TGR), and obtained stable geneticin-resistant HLHIV1-A and SVTGHIV1-A cells, respectively. The response to the retinoic acid was studied on the LTR regulated CAT activity in both cell lines. It was found that retinoic acid at a concentration of 1×10−5 effects a 3.2 - fold increase in CAT expression compared to HIV LTR in HLHIV1-A, but requires a concentration of 5×10−5 M to enhance this expression 4.6-fold in SVTGHIV1-A cells. These data show that retinoic acid may play a critical role in HIV-1 expression in human epithelial and fibroblast cell lines.

Evaluation of Western blot in routine diagnosis of hepatitis C virus

Hepatitis C virus (HCV) is among the major causes of parenterally transmitted hepatitis. Detection of infected persons would greatly diminish transmission rates and is therefore a significant parameter for prevention. Current assays are not able to resolve all cases and sometimes the results are controversial. The present study outlines problems that arise during routine testing. Two ELISA tests and three confirmatory tests were used and Polymerase Chain Reaction (PCR) data were available for some of the samples. The results of this study show that only 77.4% of samples positive for both ELISAs were confirmed as being positive. Controversial ELISA results remained controversial, depending on the confirmatory test used. PCR results, though not complete, point to the major problem of Western blot (WB) negative sera that prove positive for the viral genome and have to be excluded from screening for blood and organ donation. Since PCR cannot be used as a routine screening procedure, improvement of the routine assays is needed to minimize ambiguous results.