Tumor-relevant Genes Research Articles

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Veeriah et al., pp. 2647–2655 Apple polyphenols are known to have protective effects in colon cancer, modulated, for example, through tumor suppressor genes or molecules involved in cell proliferation. Previously, Veeriah et al. discovered that apple extract (AE) could affect the expression of chemopreventive genes in a colon carcinoma cell line. In this report, the authors wanted to see whether AE has a chemopreventative effect in nonmalignant colon cells. To do this they incubated preneoplastic human adenoma colon LT97 cells with varying concentrations of AE. The resultant RNA expression was measured using two cDNA array systems: a Superarray analysis that contained gene probes related to drug metabolism and a custom-made cDNA that contained gene probes related to metabolism, signal transduction, apoptosis, cell cycle and tumor suppression, among other functions. AE-cultured LT97 cells showed increased expression in 30 of the genes - 25 of which were related to phase II metabolism and included the detoxifying enzyme systems of the glutathione S-transferase (GST) and the UDP-glucuronosyltransferase (UGTs) families. Using the custom-made cDNA microarray, statistically significant increased expression was observed for a number of tumor-relevant genes. These included capase 10 and the tumor suppressor gene PTPRJ. However, the most notable increase was found in members of the GST and UGT families. Real-time PCR confirmed the upregulation of GSTP1, GSTT2 and GSTA4s and UGT1A1 and UGT2B7. Functional activity was also observed as AE culture increased total GST and UGT enzymatic activity. The authors concede that the concentrations of AE they use in culture need to be tested in vivo to verify their results. This study, however, shows a promising protective potential of apple polyphenols; their presence may enhance the expression of detoxifying enzymes in healthy colon tissue that exert an anti-carcinogenic effect. Bao et al., pp. 2699–2706 1α,25-Dihydroxyvitamin D3 (1,25-VD), the active form of vitamin D, is known to reduce the incidence of many human cancers, including prostate cancer. Less is known about the anti-oxidative effects of 1,25-VD. In this study, Bao et al., investigated the potential of 1,25-VD to overcome oxidative damage inflicted by reactive oxygen species (ROS) in prostate cells. To do so, the authors challenged human malignant and nonmalignant human prostate epithelial cell lines with H2O2 with and without 1,25-VD treatment. Interestingly 1,25-VD increased cell survival in nonmalignant but not in malignant cell lines. Gene expression profiling had previously revealed that 1,25-VD upregulates several genes involved in redox balance, including glucose-6-phosphatedehydrogenase (G6PD). The authors confirmed 1,25-VD upregulates G6PD — in a time- and dose-dependent manner - in nonmalignant but not in malignant prostate cells. The upregulation of G6PD led to an increase in glutathione level and a decrease in ROS activity in nonmalignant prostate cells. By using a G6PD inhibitor, the protective effects of 1,25-VD on cell survival were largely blocked. Chromatin immunoprecipitation identified a functional vitamin D response element (VDRE) in the first intron of G6PD. This study clearly shows that 1,25-VD can protect normal prostate cells against oxidative damage, an effect mediated through G6PD. Importantly, this anti-oxidative effect is dependent on the cells being in a nonmalignant state. Ju et al., pp. 2784–2790 Renal cell carcinoma (RCC) is an aggressive cancer that is resistant to standard treatments. RCC is known to contain tumor-infiltrating lymphocytes (TIL). In this study, the authors investigated whether an anti-tumor response could be elicited in mice by using an immune-mediated approach — in combination with a standard chemotherapeutic agent, 5 -flurouracil (5-FU). The authors chose the anti-4-1BB monoclonal antibody, a known costimulatory signal for T-cells, which has been shown to induce regression in other tumors. Used alone in mice injected with RCC cells, anti-4-1BB slowed tumor growth but 7 out of 8 of the mice still died. Used alone, 5-FU was not effective either. However, when 5-FU and anti-4-iBB were given in combination, 70% of the mice were tumor free by day 20, and survival rates were much higher than in mice receiving one treatment. As shown by depletion experiments, CD8+ T-cells were the primary effectors in this tumor model. The immune-mediated tumor protection resulting from combination treatment turned out to be long lasting: tumor-free mice reinjected with RCC cells 3 months later did not develop tumors. In this important study, Ju et al. have shown that anti-4-1BB, together with a sub-toxic dose of 5-FU, can effectively eradicate RCC, an otherwise treatment-resistant cancer.. Percentage survival of mice receiving anti-4-1BB and 5-FU alone or in combination.

Amplification at 7q22 Targets CDK6 in T-Cell Lymphoma.

Genomic amplification is microscopically visible extrachromosomally as double-minute chromosomes (DMIN) or intrachromosomally as homogeneously stained regions (HSR). Both DMIN and HSR comprise iterated copies of discrete genomic regions (amplicons) effecting copy number increases associated with upregulation of target genes. In cancer cells, these usually comprise oncogenes such as MYC. Cytogenetic analysis of the anaplastic large cell lymphoma (ALCL) cell line SU-DHL-1 revealed an HSR on the long arm region of chromosome 7 (7q). Detailled analysis of this region by fluorescence in situ hybridization (FISH) using tilepath BAC clones identified an amplicon corresponding to 86–95 Mb (NCBI Build 36.1) at 7q22. Expression analysis using reverse transcription (RT)-PCR of candidate oncogenes mapped within the amplicon, comprising DBF4, SRI, AKAP9, GATAD1, CDK6 and PPP1R9A, highlighted cyclin-dependent kinase 6 (CDK6) as a plausible amplification target. With reference to other ALCL cell lines, including SR-786, lacking this amplicon, SU-DHL-1 displays upregulation of CDK6 at both the RNA and protein levels as indicated by semiquantitative RT-PCR, real-time quantitative (RQ)-PCR and immuno-cytochemistry. Another ALCL cell line, L-82, containing a larger amplicon (7q21-31), also showed enhanced CDK6 expression as analyzed by RQ-PCR. For a functional test both SU-DHL-1 and SR-786 cells were treated with rapamycin, an inhibitor of the AKT-pathway which is involved in degradation of CDK-inhibitor p27. In contrast to SR-786, SU-DHL-1 cells are resistant to the effects of rapamycin on both proliferation and G1 cell cycle arrest, implying that CDK6 overexpression may confer proliferative advantage. Copy number analysis of several tumor-relevant genes using a commercial PCR-based approach (MLPA) determined a nearly 5-fold amplification of the CDK6 gene in SU-DHL-1. Interphase FISH confirmed copy number increase in SU-DHL-1 and identified CDK6 amplification in 1/77 ALCL and 4/38 peripheral T-cell lymphoma patient samples, indicating that this genetic event is relatively rare among T-cell lymphomas. Taken together, we have identified an amplicon at 7q22 in T-cell lymphoma cells targeting CDK6 which is an important cell cycle regulator and probably connected to the increased proliferative capacity of these malignant cells. Although CDK6 amplification seems to be infrequent overall, it strengthens the importance of this gene in regulating proliferation among T-cell lymphomas and may, therefore, represent a potential molecular target for therapeutic intervention in these disease entities.

Distinction of Hereditary Nonpolyposis Colorectal Cancer and Sporadic Microsatellite-Unstable Colorectal Cancer through Quantification of MLH1 Methylation by Real-time PCR

Promoter hypermethylation occurs frequently in tumors and leads to silencing of tumor-relevant genes like tumor suppressor genes. In a subset of sporadic colorectal cancers (CRC), inactivation of the mismatch repair gene MLH1 due to promoter methylation causes high level of microsatellite instability (MSI-H). MSI-H is also a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) in which mismatch repair inactivation results from germ-line mutations. For differentiation of sporadic and hereditary MSI-H tumor patients, MLH1 promoter methylation analysis is a promising tool but is not yet used in daily diagnostics because only qualitative techniques without standardization are available. The aim of this study is to establish a reliable and quantitative MLH1 methylation analysis technique and to define valid MLH1 methylation cutoff values for HNPCC diagnostics. We developed a new real-time PCR-based technique to detect and quantify methylation of both proximal and distal hMLH1 promoter regions. We established and validated this technique in a cohort of 108 CRCs [94 MSI-H and 16 microsatellite stable (MSS) cases] comprising a reference (n = 58) and a tester tumor group (n = 50). The reference tumor group contained 28 HNPCC with proven germ-line mutations or positive Amsterdam I criteria (median age, 37 years) and loss of MLH1 expression, 14 sporadic MSI-H CRC tumors with loss of MLH1 expression and BRAF V600E mutation (median age, 80.5 years), and 16 sporadic MSS CRC (median age, 76.5 years). No MLH1 promoter methylation could be found in any MSS tumors. HNPCC patients showed no or low level of MLH1 promoter methylation. A cutoff value of 18% methylation extent could be determined in this study to define MLH1 hypermethylation specific for sporadic MSI-H cases. Methylation could also be verified qualitatively by melting point analysis. BRAF V600E mutations were not detected in any HNPCC patients (n = 22 informative cases). According to the present data, quantitative MLH1 methylation analysis in MSI-H CRC is a valuable molecular tool to distinguish between HNPCC and sporadic MSI-H CRC. The detection of a BRAF V600E mutation further supports the exclusion of HNPCC.

Parathyroid Hormone-related Protein Regulates Tumor-relevant Genes in Breast Cancer Cells

The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1-34 and 67-86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin alpha6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.

Gene silencing through RNA interference (RNAi) in vivo: Strategies based on the direct application of siRNAs

RNA interference (RNAi) offers great potential not only for in vitro target validation, but also as a novel therapeutic strategy based on the highly specific and efficient silencing of a target gene, e.g. in tumor therapy. Since it relies on small interfering RNAs (siRNAs), which are the mediators of RNAi-induced specific mRNA degradation, a major issue is the delivery of therapeutically active siRNAs into the target tissue/target cells in vivo. For safety reasons, strategies based on (viral) vector delivery may be of only limited clinical use. The more desirable approach is to directly apply catalytically active siRNAs. This review highlights the recent knowledge on the guidelines for the selection of siRNAs which show high activity in the absence of non-specific siRNA effects. It then focuses on approaches to directly use siRNA molecules in vivo and gives a comprehensive overview of in vivo studies based on the direct application of siRNAs to induce RNAi. One promising approach is the in vivo siRNA delivery through complexation of chemically unmodified siRNAs with polyethylenimine (PEI). The anti-tumoral effects of PEI/siRNA-based targeting of tumor-relevant genes in vivo are described.

Automated Screening for Genomic Imbalances in Multiple Myeloma Using Microarray-Based Comparative Genomic Hybridization (mCGH).

Background: Chromosomal abnormalities are detectable in virtually all patients with multiple myeloma (MM) by the use of molecular cytogenetic techniques (e.g. fluorescence in-situ hybridization, FISH; comparative genomic hybridization, CGH). Genomic rearrangements play a crucial role in the pathogenesis of clonal plasma cell disorders and can deliver important prognostic information. Microarray-based comparative genomic hybridization (mCGH) is an innovative technique that enables a genome-wide, high-resolution tumor cell screening for chromosomal imbalances in a single experiment. The aim of the present study was the detailed characterization of genomic gains and losses in MM - in particular the delineation of critical regions - by the use of mCGH.Methods: So far, bone marrow specimens from 55 patients with Salmon &Durie stage II or III MM were analyzed. To achieve a plasma cell proportion greater than 50% before hybridization, CD 138-enrichment by immunomagnetic separation was performed whenever necessary. mCGH chips consisting of approximately 6.400 DNA clones, among them ~3.200 clones covering the whole human genome in 1 Mb increments, the remaining forming contigs from specific genomic regions or containing tumor-relevant genes were applied.Results: 52 out of 55 patients (94.5%) were evaluable by mCGH. Gains and losses of chromosomal material were found in 51 out of 52 patients (98%). In one case, mCGH did not exhibit imbalances but +9q34 and +11q25 was diagnosed by FISH for yet unknown reasons. Genomic losses most frequently involved chromosomes 13 (65%), 1p (39%), 16 (31%), 6 (27%), and 12 (21%), while gains commonly affected chromosomes 1q, 9, 11, 15 (46%), and 19 (35%). High-level amplifications were identified on chromosome 16 in two cases (overall three amplicons involving chromosome bands 16p11-p13, 16q12, and 16q21, respectively) as well as on chromosomes 8 (three amplified segments, one of them containing c-myc at 8q24) and 20q (2 amplifications involving bands 20q13.1 and 20q13.3) in one case each. Data on chromosome 13 deletion was consistent with monosomy 13 in 28 out of 34 cases (82.3%). For the remaining 6 cases with a partial 13q loss, no commonly deleted region could be delineated. In contrast, single critical regions were identified on other chromosomes, e.g. chromosome 14 (+14q23.2-q24.3). A small genomic region defined by only four closely adjacent 11q DNA clones was gained in 23 out of 24 (96%) cases with chromosome 11 extra copies.Conclusions: mCGH allows the detection of genomic imbalances including high-level amplifications in almost all patients with MM and enables a precise delineation of critical regions in this disease. Evaluation of mCGH raw data is ongoing and confirmative FISH analyses are currently under way. Supported by grants from the Deutsche José Carreras Leukämie-Stiftung (DJCLS-R04/04) and the Wilhelm Sander-Stiftung (No. 2002.098.1) to P.L.

Systematic review of genomic integration sites of human papillomavirus genomes in epithelial dysplasia and invasive cancer of the female lower genital tract.

Cancers of the anogenital tract as well as some head and neck cancers are caused by persistent infections with high-risk type human papillomaviruses (HPVs). Two viral oncogenes, E6 and E7, induce severe chromosomal instability associated with centrosome aberrations, anaphase bridges, chromosome lagging, and breaking. This occurs early in preneoplastic lesions, when the viral genome still persists in an episomal state. In most invasive cancers and also in a few high-grade dysplastic lesions, however, integration of high-risk HPV genomes into the host genome is observed. Integration seems to be a direct consequence of chromosomal instability and an important molecular event in the progression of preneoplastic lesions. Disruption or deregulation of defined critical cellular gene functions by insertional mutagenesis by integrated HPV genome fragments has been hypothesized as one major promoting factor in the pathogenesis of HPV-associated cancers. This hypothesis was based on the detection of HPV integration events in the area of tumor-relevant genes in few cases. Here, we reviewed >190 reported integration loci with respect to changes in the viral structure and the targeted genomic locus. This analysis confirms that HPV integration sites are randomly distributed over the whole genome with a clear predilection for genomic fragile sites. No evidence for targeted disruption or functional alteration of critical cellular genes by the integrated viral sequences could be found.

Tissue-wide expression profiling using cDNA subtraction and microarrays to identify tumor-specific genes.

With the objective of discovering novel putative intervention sites for anticancer therapy, we compared transcriptional profiles of breast cancer, lung squamous cell cancer (LSCC), lung adenocarcinoma (LAC), and renal cell cancer (RCC). Each of these tumor types still needs improvement in medical treatment. Our intention was to search for genes not only highly expressed in the majority of patient samples but which also exhibit very low or even absence of expression in a comprehensive panel of 16 critical (vital) normal tissues. To achieve this goal, we combined two powerful technologies, PCR-based cDNA subtraction and cDNA microarrays. Seven subtractive libraries consisting of approximately 9250 clones were established and enriched for tumor-specific transcripts. These clones, together with approximately 1750 additional tumor-relevant genes, were used for cDNA microarray preparation. Hybridizations were performed using a pool of 16 critical normal tissues as a reference in all experiments. In total, we analyzed 20 samples of breast cancer, 11 of LSCC, 11 of LAC, and 8 of RCC. To select for genes with low or even no expression in normal tissues, expression profiles of 22 different normal tissues were additionally analyzed. Importantly, this tissue-wide expression profiling allowed us to eliminate genes, which exhibit also high expression in normal tissues. Similarly, expression signatures of genes, which are derived from infiltrating cells of the immune system, were eliminated as well. Cluster analysis resulted in the identification of 527 expressed sequence tags specifically up-regulated in these tumors. Gene-wise hierarchical clustering of these clones clearly separated the different tumor types with RCC exhibiting the most homogeneous and LAC the most diverse expression profile. In addition to already known tumor-associated genes, the majority of identified genes have not yet been brought into context with tumorigenesis such as genes involved in bone matrix mineralization (OSN, OPN, and OSF-2) in lung, breast, and kidney cancer or genes controlling Ca(2+) homeostasis (RCN1,CALCA, S100 protein family). EGLN3, which recently has been shown to be involved in regulation of hypoxia-inducible factor, was found to be highly up-regulated in all RCCs and in half of the LSCCs analyzed. Furthermore, 42 genes, the expression level of which correlated with the overall survival of breast cancer patients, were identified. The gene dendogram clearly separates two groups of genes, those up-regulated such as cyclin B1, TGF-beta 3, B-Myb, Erg2, VCAM-1, and CD44 and those down-regulated such as MIG-6, Esp15, and CAK in patients with short survival time.

Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes).

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor beta stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields.

Comparative architectural aspects of regions of conserved synteny on human chromosome 11p15.3 and mouse chromosome 7 (including genes WEE1 and LMO1)

Human chromosome 11p15.3 is associated with chromosome aberrations in the Beckwith Wiedemann Syndrome and implicated in the pathogenesis of different tumor types including lung cancer and leukemias. To date, only single tumor-relevant genes with linkage to this region (e.g. LMO1) have been found suggesting that this region may harbor additional potential disease associated genes. Although this genomic area has been studied for years, the exact order of genes/chromosome markers between D11S572 and the WEE1 gene locus remained unclear. Using the FISH technique and PAC clones of the flanking markers we determined the order of the genomic markers. Based on these clones we established a PAC contig of the respective region. To analyse the chromosome area in detail the synteny of the orthologous region on distal mouse chromosome 7 was determined and a corresponding mouse clone contig established, proving the conserved order of the genes and markers in both species: “cen–WEE1–D11S2043–ZNF143–RANBP7–CEGF1– ST5–D11S932–LMO1–D11S572–TUB–tel”, with inverted order of the murine genes with respect to the telomere/centromere orientation. The region covered by these contigs comprises roughly 1.6 MB in human as well as in mouse. The genomic sequence of the two subregions (around WEE1 and LMO1) in both species was determined using a shotgun sequencing strategy. Comparative sequence analysis techniques demonstrate that the content of repetitive elements seems to decline from centromere to telomere (52.6% to 34.5%) in human and in the corresponding murine region from telomere to centromere (41.87% to 27.82%). Genomic organisation of the regions around WEE1 and LMO1 was conserved, although the length of gene regions varied between the species in an unpredictable ratio. CpG islands were found conserved in putative promoter regions of the known genes but also in regions which so far have not been described as harboring expressed sequences.

Primary leiomyosarcoma of bone: clinicopathologic, immunohistochemical, and molecular biologic aspects.

Primary leiomyosarcoma of bone is a very rare malignant tumor with uncertain pathogenicity. The authors studied five cases of surgically treated primary leiomyosarcoma of bone. Clinical histories and radiographic findings were recorded. Regular clinical and radiographic controls were obtained postoperatively. In all cases, immunohistochemical studies were used to confirm the diagnosis. Molecular biologic examinations, using the polymerase chain reaction technique with microsatellite DNA markers from regions of tumor-relevant genes, were performed to determine the stability of the genome or to detect some typical genomic changes. The study included three women and two men, with an average age of 42 years. The tumor was located in the pelvis in two patients, in the femur in two patients, and in the proximal tibia in one patient. All tumors were classified as high-grade tumors (four stage IIB, one stage IIA). Radiographically, all tumors appear as purely osteolytic lesions, with a geographic or moth-eaten appearance and without any sclerotic margin. Three patients underwent limb salvage surgery followed by endoprosthetic replacement. The other two patients required amputation. The mean follow-up was 19 months (range, 8-29 months). Three patients died of disease, with a mean postoperative survival period of 18 months (range, 6-27 months). Four patients developed diffuse pulmonary metastases after an average of 10.5 months. One of those patients responded well to chemotherapy. In all cases, immunohistochemistry showed strong reactivity of the tumor cells for (alpha-SMA and vimentin. Molecular biologic investigations revealed a high rate of genomic instabilities in all of the stage IIB tumors. Clinical follow-up suggests that primary osseous leiomyosarcoma has an aggressive biologic behavior. The immunohistochemical studies are useful tools and suggest that osseous leiomyosarcoma arise from the vascular smooth muscle cells within the bone. The molecular biologic findings of a high rate of genomic instability confirm the hypothesis that this rare entity is of an aggressive nature.

Translation initiation factor eIF-4gamma is encoded by an amplified gene and induces an immune response in squamous cell lung carcinoma.

Amplification of cellular oncogenes is an important mechanism of altered gene expression in human cancers. Using comparative genomic hybridization we recently identified an amplification at 3q26.1-q26.3 in 30% of squamous cell carcinomas of the lung. A variety of methods including microdissection-mediated procedures permit cloning of genes encoded within amplified domains but do not directly lead to the identification of biologically relevant genes. In this study, we have circumvented this problem by combining an immunological and molecular genetic approach to analyze squamous cell lung carcinoma. To identify both amplified and tumor relevant genes, we generated a cDNA expression library from a tumor with the 3q amplification and hybridized the expressed recombinant polypeptides with the autologous serum. Of 400000 cDNA clones we identified 17 antigens which induce an immune response in a patient with squamous cell lung carcinoma. While most clones represent individual genes sequence analysis revealed that four of the 17 cDNAs are nearly identical with the eukaryotic translation initiation factor (eIF)-4gamma recently assigned on 3q. We demonstrated that the gene for eIF-4gamma was amplified within 3q26-q27 in independent squamous cell lung carcinomas. In this study, we report the identification of several antigens which elicit an immune response in a squamous cell lung carcinoma patient including eIF-4gamma. eIF-4gamma is encoded by an amplified gene and possibly plays a crucial part in the development of squamous cell lung carcinoma.

DNA alterations in sporadic colorectal tumors do not correlate with tumor staging diagnosed by the TNM system.

Blood normal and tumor tissue samples of 23 patients with sporadic colorectal tumors were screened for DNA alterations in the tumor relevant genes APC, K-ras, DCC and p53. Six different microsatellite regions were analyzed for instability by a new developed non-radioactive method. Somatic DNA alterations were found in 17 tumor samples: 13 carried single or multiple changes in single genes; six carried alterations in microsatellites; two tumors showed tumor suppressor gene mutations in addition to microsatellite changes. We found no indications of correlations between current genetic models of colorectal tumor progression and the established TNM system for histopathological tumor classification.

Cancer risk and oxidative DNA damage in man.

In living cells reactive oxygen species (ROS) are formed continuously as a consequence of metabolic and other biochemical reactions as well as external factors. Some ROS have important physiological functions. Thus, antioxidant defense systems cannot provide complete protection from noxious effects of ROS. These include oxidative damage to DNA, which experimental studies in animals and in vitro have suggested are an important factor in carcinogenesis. Despite extensive repair oxidatively modified DNA is abundant in human tissues, in particular in tumors, i.e., in terms of 1-200 modified nucleosides per 10(5) intact nucleosides. The damaged nucleosides accumulate with age in both nuclear and mitochondrial DNA. The products of repair of these lesions are excreted into the urine in amounts corresponding to a damage rate of up to 10(4) modifications in each cell every day. The most abundant of these lesions, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), is also the most mutagenic, resulting in GT transversions which are frequently found in tumor relevant genes. A series of other oxidative modifications of base and sugar residues occur frequently in DNA, but they are less well studied and their biological significance less apparent. The biomarkers for study of oxidative DNA damage in humans include urinary excretion of oxidized nucleosides and bases as repair products and modifications in DNA isolated from target tissue or surrogate cells, such as lymphocytes. These biomarkers reflect the rate of damage and the balance between the damage and repair rate, respectively. By means of biomarkers a number of important factors have been studied in humans. Ionizing radiation, a carcinogenic and pure source of ROS, induced both urinary and leukocyte biomarkers of oxidative DNA damage. Tobacco smoking, another carcinogenic source of ROS, increased the oxidative DNA damage rate by 35-50% estimated from the urinary excretion of 8-oxodG, and the level of 8-oxodG in leukocytes by 20-50%. The main endogenous source of ROS, the oxygen consumption, showed a close correlation with the 8-oxodG excretion rate although moderate exercise appeared to have no immediate effect. So far, cross-sectional study of diet composition and intervention studies, including energy restriction and antioxidant supplements, have generally failed to show an influence on the oxidative DNA modification. However, a diet rich of Brussels sprouts reduced the oxidative DNA damage rate, estimated by the urinary excretion of 8-oxodG, and the intake of vitamin C was a determinant for the level of 8-oxodG in sperm DNA. A low-fat diet reduced another marker of oxidative DNA damage in leukocytes. In patients with diseases associated with a mechanistically based increased risk of cancer, including Fanconi anemia, chronic hepatitis, cystic fibrosis, and various autoimmune diseases, the biomarker studies indicate an increased rate of oxidative DNA damage or in some instances deficient repair. Human studies support the experimentally based notion of oxidative DNA damage as an important mutagenic and apparently carcinogenic factor. However, the proof of a causal relationship in humans is still lacking. This could possibly be supported by demonstration of the rate of oxidative DNA damage as an independent risk factor for cancer in a prospective study of biobank material using a nested case control design. In addition, oxidative damage may be important for the aging process, particularly with respect to mitochondrial DNA and the pathogenesis of inflammatory diseases.

The hepatitis B virus PreS2/St transactivator utilizes AP-1 and other transcription factors for transactivation

Integrated hepatitis B virus DNA cloned from hepatitis B virus-associated hepatocellular carcinoma frequently contains 3'-truncated middle surface genes (preS2/St), which were recently found to have a transcriptional transactivator function. Because preS2/St, among others, is able to transactivate the promoters of the cellular oncogenes c-myc and c-fos, it has been speculated that integrated preS2/St genes might contribute to hepatitis B virus-associated liver carcinogenesis. In this study, we investigated the mechanism of target gene stimulation by preS2/St. It was found that deletion of a fragment containing the binding site for transcription factor AP-1 (Jun-Fos) substantially decreases inducibility of the human c-myc promoter by preS2/St. A subsequent investigation of AP-1 activation by preS2/St revealed the following: (a) insertion of multimeric AP-1 binding sites confers inducibility to an otherwise unstimulatable test promoter; (b) transactivation of AP-1 sites is dramatically increased when Jun and Fos are overexpressed by cotransfected expression plasmids; and (c) inhibitors of AP-1 activation also impair transactivation by preS2/St. Besides AP-1, preS2/St was also able to utilize the unrelated transcription factors NF-kappa B and AP-2 for transactivation, suggesting that the gene product of preS2/St acts indirectly through one or several general cellular pathways rather than as a bona fide transcription factor. Because AP-1 conveys induction of a large panel of tumor-relevant genes, its preS2/St-dependent activation implies a possible causative role in hepatitis B virus-associated hepatocarcinogenesis.