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

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 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.

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.

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.

The presence of herpesviruses in pterygium.

Pterygium is a chronic disease of unknown origin and pathogenesis. It is a vision threatening disease where surgical excision is effective. We examined surgically excised symptomatic pterygia for the presence of herpesviruses such as cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) DNA using the polymerase chain reaction (PCR) technique. Samples of normal conjuctival tissue from limpus at 12 or 6 hours were excised in some of the eyes treated; they were used as controls. HSV DNA was detected in 9 and CMV DNA in 8 out of the 20 examined samples. In 3 out of the 20 examined samples both HSV and CMV DNA were detected whereas EBV DNA was not found in any of the examined samples. These results suggest that HSV and CMV may contribute to the pathogenesis of pterygium.

Binding of wild-type and mutant forms of p53 protein from human tumors to a specific DNA-sequence of the first intron of the h-ras oncogene.

p53 is the most frequent target for genetic alterations in a wide variety of human cancers. The product of the p53 tumor suppressor gene binds to DNA and activates transcription from promoters containing its consensus binding site. In the accompanying paper we have found that P53 tumor suppressor protein recognizes specifically a transcriptional element within the human H-ras protooncogene (Spandidos DA, et al, Int J Oncol 7: 1029-1034, 1995). We transfected Saos-2 cells, which are p53-null cells, with plasmids encoding for the wild type (wt) and for one 'hot spot' mutant (mt) of the p53 gene (H 273). Using the resulted nuclear extracts for gel retardation assays, we showed binding of both the wild-type and the mutant form of p53 to the H-ras DNA. Furthermore, using nuclear extracts from head and neck tumors and from adjacent normal tissues in gel retardation assays, we found binding of both the wild-type and the p53 mutant in the same responsive element of the H-ras oncogene. These experimental results suggest a direct role of p53 in regulation of H-ras. Identification of cellular proto-oncogenes as mediators of the transcriptional effects of wild-type and mutant forms of p53 gene, will be a step towards a better understanding of the role of oncogenes and once-suppressor genes in tumor promotion.

Detection of hepatitis-B virus-DNA and mutations in k-ras and p53 genes in human hepatocellular carcinomas.

Hepatitis B virus (HBV) infection is considered as one of the major risk factors in the development of human hepatocellular carcinoma (HCC). Recent studies have also suggested the implication of oncogene and onco-suppressor genes in liver carcinogenesis. We studied 41 cases of HCC for the presence of HBV DNA and point mutations in codon 12 of K-ras and codon 249 of p53. We used 'nested' PCR for the amplification of HBV because of the expected low incidence of the virus DNA in the samples. PCR was also used for the amplification of K-ras and p53 regions that contain the codons of interest, followed by RFLP analysis for the detection of point mutations. HBV DNA was amplified in 22 cases (53.7%), while 5 cases (12.2%) appeared to carry mutations in codon 12 of K-ras and 7 cases (17.1%) had mutations in codon 249 of the p53 gene. These results further support the correlation between HBV infection and HCC and also indicate an implication of K-ras and p53 genes in hepatocarcinogenesis.

Detection and analysis of hepatitis C virus by a combined RT-PCR method: variation in the 5' non-coding region of the viral genome.

A combined reverse transcription-polymerase chain reaction (RT-PCR) method was employed for the detection of hepatitis C virus (HCV) RNA in serum from patients with chronic active hepatitis, with primers corresponding to the 5' non-coding region. The diagnosis was based on serological and biochemical methods and on liver biopsy. HCV-RNA was detected in 27 (90%) of 30 sera examined. The nucleotide sequence of PCR-amplified HCV cDNAs (256 bp) was determined from five specimens and heterogeneity varying between 0.58% and 2.89% among the clinical samples and the prototype HCV-1 was found.

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.

Ap-1 recognizes sequence elements on hiv-1 LTR in human epithelial tumor-cell lines.

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-L protein in the regulation of HIV-1 expression. The breast MDA MB 468 and cervical HeLa turner 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.

Detection of herpesvirus-like DNA sequences in Mediterranean Kaposi's sarcoma.

The aetiology of Kaposi's sarcoma remains obscure, however, epidemiological studies indicate that the disease possesses an infectious aetiology. Recent data revealed the presence of specific herpesvirus-like DNA sequences (KHSV) in all forms of Kaposi's sarcoma indicating that a novel virus may be the infectious agent which causes the disease. The aim of the present investigation was to assess the incidence of this herpesvirus-like DNA sequence in 28 Mediterranean Kaposi's sarcomas. DNA was extracted from formalin-fixed paraffin-embedded tissues and analysed by a sensitive PCR based assay. The KSHV specific DNA sequences were found in 22 of 28 (79%) cases suggesting a potential important role in the development of the disease.

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.

Detection of the epstein-barr-virus by the polymerase chain-reaction in immunosuppressed and immunocompromised patients.

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 B 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.

Detection of hepatitis C virus in sera and genotyping according to the 5' non-coding region.

A reverse transcription (RT)-polymerase chain reaction (PCR) method was used for detection of the RNA of hepatitis C virus (HCV) in 120 samples of sera from Crete, which were positive for HCV-specific antibodies, by ELISA and Western blot analyses. A segment of 255 bp, located in the most conserved region of the HCV genome (the 5' untranslated region, 5' UTR), was amplified. For the identification of sequence variation from the HCV-1 strain, twenty of these samples were sequenced and compared to prototype strain (HCV-1) according to current genotypic classification. We were able to identify fourteen of the twenty as type 1a (i.e. similar to the prototype), two as type 1b, two as type 3a and two as type 4a. These findings generally agree with the geographic distribution of the already identified genotypes, though 3a type has not been reported previously in Crete (Greece).

Activating mutations of ras family genes in prostatic-cancer.

ras family genes (H-, K- and N-ras) encode for a 21 kD membrane protein which possesses GTPase activity and participates in a signal transduction pathway. Activating mutations of the ras family genes occur at codons 12, 13 and 61 and have been detected in a variety of human tumours, including colonic, bladder and pancreatic cancers. Prostatic cancer is among the most common malignancies throughout the world and a major cause of death from cancer in males. Data reported on the implication of the ras family genes in the development of the disease are conflicting. The aim of this study was to determine the incidence of mutations at codon 12 of H-ras, codon 12 of K-ras and codon 61 of N-ras proto-oncogenes, in a Greek population with prostatic cancer. Our analysis revealed that 4 out of 20 (20%) samples harboured a K-ras codon 12 point mutation, 1 out of 20 (5%) specimens contained mutations at codon 12 of the H-ras and 1 out of 20 (5%) at codon 61 of the N-ras, indicating a role for the ras genes in the development of the disease.

Detection of activating mutations in the ras family genes in cytological specimens from lung-tumors.

Mutations in the ras family genes (K-ras mainly) represent a common event in lung tumorigenesis which is frequently associated with poor clinical outcome. In order to investigate whether K-ras mutations are detectable in cytological material obtained from patients with lung cancer, 37 cytological specimens (16 fine needle aspiration and 21 bronchoscopy) were assessed for codon 12 point mutations in the H-, K- and N-ras genes by combined polymerase chain reaction-restriction fragment length polymorphism. K-ras codon 12 point mutations were found in 8 out of 37 (22%) specimens while no mutations were found in the H-ras and N-ras genes. Mutations were found in 27% (3 out of 11) of adenocarcinomas while in squamous cell carcinomas the incidence of mutations was 18% (3 out of 17). In addition, a K-ras codon 12 point mutation was found in one (12%) among 8 small cell carcinomas and in the only Hodgkin's lymphoma with metastasis in the lung. Our results are in agreement with previous results that recognise high incidence of K-ras activation in lung carcinomas, and indicate that detection of mutant ras alleles is possible in cytological material.

Instability at microsatellite sequences in spontaneously aborted human embryos provides evidence for a novel mechanism for recurrent miscarriages.

Several factors have been proposed to confer a risk for abortion of the embryo. However, the aetiology of spontaneous abortions remains unclear. In the present study we investigated if an increased mutational rate occurs in the embryonic tissue and whether this phenomenon is associated with recurrent miscarriage. The mutational rate was assessed in 30 spontaneously aborted embryos using a bank of 8 highly polymorphic microsatellite markers, each one located on a different chromosome. The microsatellite sequences of DNA extracted from distal sites of each embryo were amplified by the polymerase chain reaction and the electrophoretic patterns were compared. Shifts in the mobility of the microsatellites indicating instability were scored for 12 among 30 (40%) specimens, thus suggesting that microsatellite instability (MI) is a relatively common feature of spontaneously aborted embryonic tissues. Association was found between instability and the absence of normal childbirth: 11 among 18 cases without a normal childbirth exhibited evidence of MI while only one among 12 cases with normal childbirth was positive for MI. Our results suggest that instability at microsatellite sequences which indicate decreased fidelity in DNA replication and repair are associated with the recurrent abortion of the embryo, particularly in cases without a normal childbirth.

Microsatellite instability in patients with chronic obstructive pulmonary disease.

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.

TGF-beta 1 overexpression in breast cancer.

TGF-beta 1 belongs to a family of pluripotent growth factors (TGF beta s) and has been implicated in the development and progression of human breast cancer. There are conflicting data though, suggesting that TGF-beta has the pontency both to promote and inhibit the progression of mammary neoplasia. We examined the expression of TGF-beta 1 mRNA in 24 breast carcinomas using the technique of the reverse transcription polymerase chain reaction (RT-PCR) to obtain quantitative results. Overexpression of TGF-beta 1 gene was found in 75% of the cases. We also correlated the overexpression of the TGF-beta 1 gene with clinicopathological parameters including histological grade, tumour cellularity, oestrogen receptor status (ER), progesterone receptor status (PR) and lymph node involvement. The results led us to the conclusion that the increasing ratio of overexpression related to the stage of cancer in an analogous way (P similar to 1). No significant association was identified between the ratio of overexpression and the grade, ER, PR, or lymph node involvement (r(s) = 0.5, 0.2, 0.1, 0.1 respectively; P < 0.0001) in all categories.

Detection of human cytomegalovirus and epstein-barr-virus by the polymerase chain-reaction in patients with Beta-thalassemia.

Infections caused by Human Cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) are common in multiple transfused patients, such as patients with beta-thalassaemia. The ability of the Polymerase Chain Reaction (PCR) to amplify HCMV and EBV DNA from blood and other samples makes this technique a valuable diagnostic tool for the detection of both viruses in the early stages of the infection. PCR was used for the amplification of a 435 bp region of the immediate early-1 (IE-1) gene of HCMV and a 375 bp sequence from the EcoRI B fragment of EBV genome. Blood samples from 80 patients with beta-thalassaemia were examined. HCMV was found in 14 and EBV in 12 patients. The results obtained confirm the implications of HCMV and EBV in the diagnosis of viral infections in multiple transfused patients as well as the importance of PCR technique as a valuable diagnostic tool.