Telomeric repeats on small polydisperse circular DNA (spcDNA) and genomic instability.
(25/9730)
Small polydisperse circular DNA (spcDNA) is a heterogeneous population of extrachromosomal circular molecules present in a large variety of eukaryotic cells. Elevated amounts of total spcDNA are related to endogenous and induced genomic instability in rodent and human cells. We suggested spcDNA as a novel marker for genomic instability, and speculated that spcDNA might serve as a mutator. In this study, we examine the presence of telomeric sequences on spcDNA. We report for the first time the appearance of telomeric repeats in spcDNA molecules (tel-spcDNA) in rodent and human cells. Restriction enzyme analysis indicates that tel-spcDNA molecules harbor mostly, if not exclusively, telomeric repeats. In rodent cells, tel-spcDNA levels are higher in transformed than in normal cells and are enhanced by treatment with carcinogen. Tel-spcDNA is also detected in some human tumors and cell lines, but not in others. We suggest, that its levels in human cells may be primarily related to the amount of the chromosomal telomeric sequences. Tel-spcDNA may serve as a unique mutator, through specific mechanisms related to the telomeric repeats, which distinguish it from the total heterogeneous spcDNA population. It may affect telomere dynamics and genomic instability by clastogenic events, alterations of telomere size and sequestration of telomeric proteins. (+info)
Increased activator protein-1 DNA binding and c-Jun NH2-terminal kinase activity in human breast tumors with acquired tamoxifen resistance.
(26/9730)
Human breast tumors that are initially responsive to tamoxifen (TAM) eventually relapse during treatment. Estrogen receptor (ER) expression and function are often preserved in these tumors, and clinical evidence suggests that this relapse may be related to TAM's known agonistic properties. ER can interact with the activator protein-1 (AP-1) transcription factor complex through protein-protein interactions that are independent of ER DNA binding and, in certain ER-positive cells, this may allow TAM to exert an agonist response on AP-1-regulated genes. We, therefore, assessed both AP-1 DNA binding and the known AP-1 activating enzyme, c-Jun NH2-terminal kinase (JNK), in a panel of 30 ER-positive primary human breast tumors with acquired TAM resistance, as compared to a matched panel of 27 untreated control ER-positive breast tumors and a separate control set of 14 primary tumors, which included 7 ER-positive tumors that were growth-arrested by 3 months of preoperative TAM. AP-1 DNA binding activity was measured from cryopreserved tumor extracts using a labeled oligonucleotide probe containing a consensus AP-1 response element by electrophoretic mobility shift assay. JNK was first extracted from the tumor lysates by incubation over a Sepharose-bound c-Jun(1-89) fusion protein, and its activity was then measured by chemiluminescent Western blot by detection of the phosphorylated product using a phospho-Jun(Ser-63)-specific primary antibody. The set of control ER-positive breast tumors growth arrested by TAM showed no significant difference from untreated control tumors in their AP-1 DNA binding and JNK activities. In contrast, there was a significant (P < 0.001) increase in mean AP-1 DNA binding activity for the panel of ER-positive TAM-resistant (TAM-R) tumors as compared to its matched control panel of untreated tumors. Mean JNK activity in the TAM-R tumors was also significantly higher than that found in the untreated tumors (P = 0.038). Overall, there was no significant correlation between JNK activity and AP-1 DNA binding; however, regression analysis showed that, for any given level of JNK activity, the TAM-R tumors possessed a 3.5-fold increase in AP-1 DNA binding activity as compared to the untreated tumors. These findings indicate that, when compared to untreated ER-positive primary breast tumors, TAM-R tumors demonstrate significantly increased levels of AP-1 DNA binding and JNK activity, consistent with experimental models suggesting that TAM-stimulated ER-positive tumor growth may be mediated by enhanced AP-1 transcriptional activity. These observations support the need for further evaluation of these markers in breast tumors as predictors of TAM resistance. (+info)
Expression of MAGE and GAGE in high-grade brain tumors: a potential target for specific immunotherapy and diagnostic markers.
(27/9730)
The mRNA expression of the tumor-associated antigens MAGE and GAGE was examined in 60 high-grade brain tumors. This analysis was performed by using reverse transcription-PCR, Southern blotting, and sequencing. It was demonstrated that, of the eight GAGE genes, GAGE-2 and -7 were expressed in five of seven normal brains. Four groups of tumors--adult glioblastoma multiforme (n = 20), pediatric glioblastoma multiforme (n = 9), medulloblastomas (n = 15), and ependymomas (n = 14)--were analyzed for mRNA expression. The following frequencies were observed: MAGE-1, 0, 0, 13, and 0%, respectively; MAGE-2, 5, 11, 60, and 57%; MAGE-3 & -6, 0, 0, 13, and 0%; GAGE-1, 65, 11, 13, and 43%; and GAGE-3-6 and -8: 75, 78, 47, and 93%, respectively. Two unclassified tumors expressed GAGE-3-6 and -8 only. The absence of GAGE-1 expression in normal brain, its relatively high frequency of expression in high-grade brain tumors, and its unique 3' sequence, suggest it may represent a useful target for specific immunotherapy. The detection method of reverse transcription-PCR and Southern blotting may also be useful for rapid screening of biopsy specimens both for diagnostic purposes and to determine a patient's eligibility for specific immunotherapy. (+info)
Increased sensitivity of hydroxyurea-resistant leukemic cells to gemcitabine.
(28/9730)
Tumor cell resistance to certain chemotherapeutic agents may result in cross-resistance to related antineoplastic agents. To study cross-resistance among inhibitors of ribonucleotide reductase, we developed hydroxyurea-resistant (HU-R) CCRF-CEM cells. These cells were 6-fold more resistant to hydroxyurea than the parent hydroxyurea-sensitive (HU-S) cell line and displayed an increase in the mRNA and protein of the R2 subunit of ribonucleotide reductase. We examined whether HU-R cells were cross-resistant to gemcitabine, a drug that blocks cell proliferation by inhibiting ribonucleotide reductase and incorporating itself into DNA. Contrary to our expectation, HU-R cells had an increased sensitivity to gemcitabine. The IC50 of gemcitabine was 0.061 +/- 0.03 microM for HU-R cells versus 0.16 +/- 0.02 microM for HU-S cells (P = 0.005). The cellular uptake of [3H]gemcitabine and its incorporation into DNA were increased in HU-R cells. Over an 18-h incubation with radiolabeled gemcitabine (0.25 microM), gemcitabine uptake was 286 +/- 37.3 fmol/10(6) cells for HU-R cells and 128 +/- 8.8 fmol/10(6) cells for HU-S cells (P = 0.03). The incorporation of gemcitabine into DNA was 75 +/- 6.7 fmol/10(6) cells for HU-R cells versus 22 +/- 0.6 fmol/10(6) cells for HU-S cells (P < 0.02). Our studies suggest that the increased sensitivity of HU-R cells to gemcitabine results from increased drug uptake by these cells. This, in turn, favors the incorporation of gemcitabine into DNA, resulting in enhanced cytotoxicity. The increased sensitivity of malignant cells to gemcitabine after the development of hydroxyurea resistance may be relevant to the design of chemotherapeutic trials with these drugs. (+info)
Association between nonrandom X-chromosome inactivation and BRCA1 mutation in germline DNA of patients with ovarian cancer.
(29/9730)
BACKGROUND: Most human female cells contain two X chromosomes, only one of which is active. The process of X-chromosome inactivation, which occurs early in development, is usually random, producing tissues with equal mixtures of cells having active X chromosomes of either maternal or paternal origin. However, nonrandom inactivation may occur in a subset of females. If a tumor suppressor gene were located on the X chromosome and if females with a germline mutation in one copy of that suppressor gene experienced nonrandom X-chromosome inactivation, then some or all of the tissues of such women might lack the wild-type suppressor gene function. This scenario could represent a previously unrecognized mechanism for development of hereditary cancers. We investigated whether such a mechanism might contribute to the development of hereditary ovarian cancers. METHODS: Patterns of X-chromosome inactivation were determined by means of polymerase chain reaction amplification of the CAG-nucleotide repeat of the androgen receptor (AR) gene after methylation-sensitive restriction endonuclease digestion of blood mononuclear cell DNA from patients with invasive (n = 213) or borderline (n = 44) ovarian cancer and control subjects without a personal or family history of cancer (n = 50). BRCA1 gene status was determined by means of single-strand conformational polymorphism analysis and DNA sequencing. All statistical tests were two-sided. RESULTS AND CONCLUSIONS: Among individuals informative for the AR locus, nonrandom X-chromosome inactivation was found in the DNA of 53% of those with invasive cancer versus 28% of those with borderline cancer (P = .005) and 33% of healthy control subjects (P = .016). Nonrandom X-chromosome inactivation can be a heritable trait. Nine of 11 AR-informative carriers of germline BRCA1 mutations demonstrated nonrandom X-chromosome inactivation (.0002 < P < .008, for simultaneous occurrence of both). IMPLICATIONS: Nonrandom X-chromosome inactivation may be a predisposing factor for the development of invasive, but not borderline, ovarian cancer. (+info)
Immunoglobulin VH gene expression among extranodal marginal zone B-cell lymphomas of the ocular adnexa.
(30/9730)
PURPOSE: Most lymphomas of the ocular adnexa are primary extranodal non-Hodgkin's lymphomas of the B-cell type, with the most common lymphoma subtype being the extranodal marginal-zone B-cell lymphoma (EMZL). Analysis of somatic mutations in the variable (V) region of the Ig heavy (H)-chain gene segment suggests that EMZL development in other locations is dependent on antigen stimulation. The purpose of this study was to analyze the presence of somatic hypermutations in clonally rearranged Ig H-chain V genes of this lymphoma entity in the ocular adnexa and to estimate whether the mutation pattern is compatible with antigen selection. METHODS: Twenty-six cases of EMZL of the ocular adnexa were diagnosed on the basis of morphology, histology, and immunohistology. A nested polymerase chain reaction (PCR) was performed on DNA extracted from paraffin sections. The isolated PCR products were sequenced and compared with published VH germline segments to determine the number of somatic mutations in the complementarity-determining region (CDR) 2 and framework (FW) region 3. RESULTS: The number of somatic mutations in the cases of EMZL varied between 0 and 24: Five cases involved 0 to 3 somatic mutations, and the remaining 21 cases involved 4 to 24 mutations. Based on the ratio of replacement (R) to silent (S) mutations in the CDR2 or FW3 regions, antigen selection seems to have occurred in 60% of ocular adnexal EMZL. The VH3 family was the most commonly expressed germline VH family (54%), followed by VH4 (23%), with biased usage of the latter. Some germline VH1 genes used included DP-8, DP-10, DP-53, DP-63 (VH4.21), and DP-49, which are frequently used by autoantibodies (e.g., rheumatoid factors) and natural autoantibodies. CONCLUSIONS: EMZLs of the ocular adnexa have an Ig H-chain mutation pattern that supports the concept that they represent a clonal expansion of post-germinal-center memory B-cells in most instances. In two thirds of cases, antigen selection may have occurred, and autoantibodies may have a role in their development. (+info)
Analysis of p16 (CDKN2/MTS-1/INK4A) alterations in primary sporadic uveal melanoma.
(31/9730)
PURPOSE: To define more clearly the role of the tumor suppressor gene p16 in uveal melanoma by determining the relative contribution of all known mechanisms of p16 inactivation in this tumor. METHODS: A comprehensive genetic analysis of the p16 gene was performed in 33 primary sporadic ciliochoroidal and choroidal melanomas. Fourteen highly polymorphic microsatellite markers surrounding the p16 locus on chromosome 9p21 were used for the microsatellite analysis. Sequence analysis of the p16 gene was performed on those tumors with 9p21 loss of heterozygosity. To investigate methylation as an alternative mechanism of inactivation of p16, methylation-specific polymerase chain reaction was performed on all tumor DNA samples. RESULTS: Loss of heterozygosity (LOH) was found in 8 of 33 (24%) uveal melanomas. No evidence of a second region of LOH that did not include the p16 locus was found. Four cases had hemizygous losses including markers both distal and proximal to p16. Homozygous deletion of the p16 gene was detected in the 4 remaining cases by microsatellite analysis. Sequence analysis revealed no p16 mutations in the tumors with hemizygous loss of p16. Methylation of the 5' CpG island of p16 was found in one tumor with 9p21 LOH and in another without LOH. CONCLUSIONS: p16 inactivation by HD or methylation occurs in 27% of uveal melanomas, representing the most common molecular genetic alteration identified thus far in uveal melanoma. (+info)
Methylation of the ABL1 promoter in chronic myelogenous leukemia: lack of prognostic significance.
(32/9730)
The BCR-ABL chromosomal translocation is a central event in the pathogenesis of chronic myelogenous leukemia (CML). One of the ABL1 promoters (Pa) and the coding region of the gene are usually translocated intact to the BCR locus, but the translocated promoter appears to be silent in most cases. Recently, hypermethylation of Pa was demonstrated in CML and was proposed to mark advanced stages of the disease. To study this issue, we measured Pa methylation in CML using Southern blot analysis. Of 110 evaluable samples, 23 (21%) had no methylation, 17 (15%) had minimal (<15%) methylation, 12 (11%) had moderate methylation (15% to 25%), and 58 (53%) had high levels of methylation (>25%) at the ABL1 locus. High methylation was more frequent in advanced cases of CML. Among the 76 evaluable patients in early chronic phase (ECP), a major cytogenetic response with interferon-based therapy was observed in 14 of 34 patients with high methylation compared with 19 of 42 among the others (41% v 45%; P value not significant). At a median follow-up of 7 years, there was no significant difference in survival by ABL1 methylation category. Among patients who achieved a major cytogenetic response, low levels of methylation were associated with a trend towards improved survival, but this trend did not reach statistical significance. Thus, Pa methylation in CML is associated with disease progression but does not appear to predict for survival or response to interferon-based therapy. (+info)