Loss of heterozygosity and microsatellite instability in human non-neoplastic hepatic lesions.

AIMS/BACKGROUND: Carcinogenesis is thought to be a multistage process that occurs as a result of mutations in oncogenes and tumor suppressor genes. One way to monitor a vast range of these changes is by microsatellite PCR amplification that detects loss of heterozygosity and microsatellite instability between normal and tumor specimens of the same subject. Viral cirrhosis is considered a strong predisposing factor for the development of liver cancer. The aim of the study therefore was to examine precancerous hepatic lesions and compare them with others not considered as high risk for hepatocellular carcinoma. METHODS: We examined 43 subjects for 19 microsatellite markers spanning chromosomes 1, 9 and 17. Normal specimens were blood samples that were compared to liver needle biopsies. Samples were classified according to histological features as non-cancerous (10 cases) and pre-cancerous (33 cases, chronic hepatitis and cirrhosis). RESULTS: Our results indicate that there is a tendency of increased chromosomal alteration as lesions become chronic. Samples from patients with antibodies to antibodies for hepatitis C virus show more alterations than hepatitis B positive samples. Steatohepatitis, a disease of unknown etiology, appears to have a high number of microsatellite abnormalities. CONCLUSIONS: Microsatellite APOA2 located on chromosome 1, shows a statistically significant increase in the rate of loss of heterozygosity as liver lesions become more severe, indicating the presence of tumor suppressor genes which may be involved in the development of these lesions.

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 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).

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.

Mutations and expression of the ras family genes in leukemias.

The levels of expression and the incidence of codon 12 point mutations 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 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.

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.

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.

Loss of heterozygosity and microsatellite instability in human atherosclerotic plaques.

Several lines of evidence suggest that mutation events may be involved in the development of atherosclerosis. The aim of the present investigation was to perform an allelotype analysis in 30 atherosclerotic lesions in order to reveal any deletions involved in the development of the disease. Eighteen chromosomal arms were tested by one microsatellite marker located on each arm and allelic imbalance in at least one marker was observed in 7 (23%) cases. Furthermore, the analysis revealed the presence of microsatellite instability (MI) in 10 (33%) cases, suggesting that an increase in the mutation rate may be involved in the formation of the plaque. These results highlight the mutation concept for the atherogenesis and suggest that LOH and MI may be involved 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 1x10(-5) effects a 3.2 -fold increase in CAT expression compared to HIV LTR in HLHIV1-A, but requires a concentration of 5x10(-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.

Microsatellite instability in human atherosclerotic plaques.

The aetiopathology of atherosclerosis remains obscure. Although histologically the accumulation of lipids and the proliferation of the smooth muscle cells represents the main feature of the disease, little is known as regards the molecular alterations associated with the atherosclerotic lesions. In the present study we investigated whether an elevated mutational rate is detectable in human atheromatous plaques. Thirty specimens were assessed for microsatellite instability (MI) by 7 microsatellite markers and MI, in at least one marker, was apparent in 6 (20%) cases. Our data suggest that decreased fidelity in DNA replication and repair may be associated with the development of the disease.

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 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.

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 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 Burkitt's 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.

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.

Instability at the H-ras minisatellite is associated with the spontaneous abortion of the embryo.

Recently we have shown that microsatellite instability (MI) is a detectable phenomenon in aborted embryonic tissues. In the present study we investigated if instability is also detectable in a minisatellite located at the 3'-end of the H-ras proto-oncogene, affecting either the repetition number of the 28-bp core generating larger or smaller alleles or its sequence creating a detectable restriction fragment length polymorphism (RFLP). Among 30 aborted embryonic tissues, alterations at the repetition number of the core were found in 3 (10%) while point mutations were detected in 7 (23%) cases. These results indicate that structural alterations of the H-ras minisatellite may be associated with the rejection of the embryo.

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.

Ras oncogene activation in benign and malignant colorectal tumours.

Activation of ras family genes has been implicated in colorectal tumourigenesis. K-ras is the most frequently altered gene in colorectal neoplasias. The major activating mechanism involves point mutations, although recent data have shown a more complex role, through qualitative alterations. The incidence of codon 12 mutations in K-ras and N-ras genes in patients with primary colorectal adenocarcinomas was examined. We employed a non radioactive PCR-RFLP assay for the detection of mutant samples. We found a significant K-ras activation at codon 12 (38%), while the incidence of N-ras codon 12 activation was limited to 1.5%. These results are in agreement with previous reports. Association has been investigated with clinical and histopathological parameters. Point mutations appear to be more frequent in carcinomas with elements indicating a development from adenoma, in ages below 50 years, in females who had the tumor located at the rest of the large bowel in comparison with rectosigmoid and in higher grade of differentiation.