Intra-osseous ganglion of the proximal humerus: a case report.

We report a 72-year-old woman with a type-1 intra-osseous ganglion in the proximal humerus, extending to the bone surface. We conducted a systemic review of intra-osseous ganglion cases in Japan to identify clinical features and pathogenesis of this condition. The anatomical distribution between intra-osseous ganglia without a communicating soft tissue ganglion (type 1) and those with (type 2) is different. The origins of intra-osseous ganglia vary and depend on their anatomical location. They can arise from within the bone or in the adjacent soft tissue, and can progress to a type-2 lesion in either an outside-in or inside-out fashion.

Tsix-deficient X chromosome does not undergo inactivation in the embryonic lineage in males: implications for Tsix-independent silencing of Xist.

Differential induction of the X-linked non-coding Xist gene is a key event in the process of X inactivation occurring in female mammalian embryos. Xist is negatively regulated in cis by its antisense gene Tsix through modification of the chromatin structure. The maternal Xist allele, which is normally silent in the extraembryonic lineages, is ectopically activated when Tsix is disrupted on the same chromosome, and subsequently the maternal X chromosome undergoes inactivation in the extraembryonic lineages even in males. However, it is still unknown whether the single Tsix-deficient X chromosome (XDeltaTsix) in males is also inactivated in the embryonic lineage. Here, we show that both male and female embryos carrying a maternally derived XDeltaTsix could survive if the extraembryonic tissues were complemented by wild-type tetraploid cells. In addition, Xist on the XDeltaTsix was properly silenced and methylated at CpG sites in adult male somatic cells. These results indicate that the embryonic lethality caused by the maternal XDeltaTsix is solely attributable to the defects in the extraembryonic lineages. XDeltaTsix does not seem to undergo inactivation in the embryonic lineage in males, suggesting the presence of a Tsix-independent silencing mechanism for Xist in the embryonic lineage.

X-irradiation induces up-regulation of ATM gene expression in wild-type lymphoblastoid cell lines, but not in their heterozygous or homozygous ataxia-telangiectasia counterparts.

Ataxia-telangiectasia (AT) is an autosomal recessive disease. The relevant gene has been cloned and designated ATM. We studied the expression of both ATM mRNA and the ATM protein in unirradiated and X-irradiated EBV (Epstein-Barr virus)-transformed lymphoblastoid cell lines (LCLs) derived from donors who were normal (ATM + / + ), AT heterozygotes (ATM + / - ), or AT homozygotes (ATM - / - ), respectively. In ATM + / + LCLs, the levels of ATM mRNA were found to have increased by approximately 1.5-fold within 1 h of exposure to 10 Gy of X-rays, while the ATM protein levels had increased by 1.5- to 2.0-fold within 2 to 3 h of irradiation. The wild-type mRNA and protein levels both returned to their basal values fairly quickly after this time. The results obtained with the ATM + / - LCLs were quite different, however: neither the mRNA nor protein levels were found to have increased as a consequence of X-irradiation in any ATM + / - LCL. Twelve of the mutations in the ATM - / - LCLs we used were truncating mutations, and we suspected that the corresponding truncated ATM proteins would be too labile to be detected by western blot analysis. However, five of the ATM - / - LCLs produced mutant ATM proteins that were identical in molecular weight to the wild-type ATM protein. When cells from three of these five clones were exposed to X-rays, transcription of the mutant ATM genes appeared to reduce somewhat, as were the levels of protein being produced. These results suggest that the normal ATM gene responds to ionizing radiation by up-regulating its activity, whereas none of the mutant ATM genes we studied were able to respond in this way.

Enhanced phosphorylation of p53 serine 18 following DNA damage in DNA-dependent protein kinase catalytic subunit-deficient cells.

DNA-dependent protein kinase (DNA-PK) controls signal transduction following DNA damage. However, the molecular mechanism of the signal transduction has been elusive. A number of candidates for substrates of DNA-PK have been reported on the basis of the in vitro assay system. In particular, the Ser-15 amino acid residue in p53 was one of the first such in vitro substrates to be described, and it has drawn considerable attention due to its biological significance. Moreover, p53 Ser-15 is a site that has been shown to be phosphorylated in response to DNA damage. In addition, crucial evidence indicating that DNA-PK controls the transactivation of p53 following DNA damage was reported quite recently. To clarify these important issues, we conducted the experiments with dna-pkcs null mutant cells, including gene knockout cells. As a result, we detected enhanced phosphorylation of p53 Ser-18, which corresponds to Ser-15 of human p53, and significant expression of p21 and mdm2 following ionizing radiation. Furthermore, we identified a missense point mutation in the p53 DNA-binding motif region in SCGR11 cells, which were established from severe combined immunodeficient (SCID) mice and used for previous study on the role of DNA-PK in p53 transactivation. Our observation clearly indicates that DNA-PK catalytic subunit does not phosphorylate p53 Ser-18 in vivo or control the transactivation of p53 in response to DNA damage, and these results further emphasize the different pathways in which ataxia telangiectasia-mutated (ATM) and DNA-PK operate following radiation damage.

Differential cytotoxicity of clinically important camptothecin derivatives in P-glycoprotein-overexpressing cell lines.

UNLABELLED: Camptothecin and its derivatives are specific inhibitors of eukaryotic topoisomerase I (top1) and are active in cancer patients against a variety of refractory solid tumors and leukemia. PURPOSE: The present study further investigated the relationship between multidrug resistance (MDR) mediated by P-glycoproteinMDR and potential resistance to camptothecin derivatives using two experimental systems: (1) MDR KB-V1 cells selected for vinblastine resistance, and (2) NIH3T3 cells transfected with a plasmid expressing wildtype P-glycoproteinMDR multidrug transporter (NIH-MDR-G185). RESULTS: We found that both KBV-1 and NIH-MDR-G185 cells were resistant to topotecan, and that topotecan-induced cleavable complexes were reduced in KB-V1 cells, consistent with a role of P-glycoproteinMDR in cellular resistance to topotecan. By contrast, no significant resistance to camptothecin, 9-aminocamptothecin, 10, 11-methylenedioxycamptothecin, or SN-38 (the active metabolite of CPT-11) was observed in NIH-MDR-G185 cells, while KB-V1 cells were cross-resistant to these compounds but produced cleavable complexes similar to those produced by parental KB-3-1 cells. CONCLUSIONS: These results suggest that topotecan is the only camptothecin tested with significant susceptibility to MDR in cell culture, and that multidrug resistant cells such as KBV1 probably exhibit additional resistance mechanisms to camptothecins besides P-glycoproteinMDR overexpression.

Acquired camptothecin resistance of human breast cancer MCF-7/C4 cells with normal topoisomerase I and elevated DNA repair.

A camptothecin (CPT)-resistant cell line (MCF-7/C4) was established from MCF-7 cells by mutagenic treatment with methylmethanesulfonate and selection with CPT. MCF-7/C4 is 30-fold resistant to CPT and is cross-resistant to UV and cis-dichlorodiammineplatinum(II) but not to VP-16 or ionizing radiation. Topoisomerase I (top1)-mediated cleavable complexes in the presence of CPT, measured by oligonucleotide assay and by alkaline elution, were similar in both cell lines. Other top1 parameters such as top1 protein, RNA levels, and DNA relaxation were also similar in both cell lines. Thus, CPT resistance is not due to alterations in top1 activity but is caused by changes in the downstream pathways from the top1-induced damage. Both cell lines had similar doubling time (22 hr), but MCF-7/C4 cells showed reduced S-phase fraction in the absence of CPT and reduced G2 delay after CPT treatment. p53, GADD45, and p21WAF1/CIP1 were induced similarly by CPT in both cell lines. The overall repair capacity estimated by the ability of cells to reactivate UV-damaged pSV-CAT plasmid was increased in MCF-7/C4 cells. These observations suggest that enhanced DNA repair is one of the factors involved in CPT resistance.

Correlations between S and G2 arrest and the cytotoxicity of camptothecin in human colon carcinoma cells.

Previous cell line comparisons indicated that neither S-phase fraction nor topoisomerase I (top1) levels are sufficient to predict camptothecin (CPT) cytotoxicity (F. Goldwasser el al., Cancer Res., 55: 2116-2121, 1995.). To identify new determinants for CPT activity, two mutant p53 human colon cancer cell lines, SW620 and KM12, that were previously reported to have similar top1 levels and differential sensitivity to CPT were studied. No difference in the kinetics of top1-mediated DNA single-strand breaks or DNA synthesis inhibition were observed after 1 h exposure to 1 microM CPT. Pulse-labeling alkaline elution showed deficiency of damaged replicon elongation in the more sensitive SW620 cells. Consistentiy, flow cytometry analyses showed that KM12 was arrested in G2, whereas SW620 cells were irreversibly blocked in S phase. Aphidicolin protection was minimal in KM12 and more pronounced in the more sensitive SW620 cells. Thus, CPT appears to have two cytotoxic mechanisms, one protectable by aphidicolin and present in SW620 and the other not protectable by aphidicolin and common to both cell lines. SW620 exhibited also a greater capacity to break through the G2 checkpoint after DNA damage. Consistently, SW620 cells failed to down-regulate cyclin B-cdc2 kinase activity, whereas KM12 cells down-regulated cyclin B/cdc2 kinase activity within 30 min to 20 % of control level after CPT treatment. Analysis of the 7 human colon carcinoma cell lines of the NCI Anticancer Drug Screen showed that defects in replicon elongation and G2 breakthrough capability correlate with sensitivity to CPT. Our results suggest that misrepair of damaged replicons and/or alterations in DNA damage checkpoints is critical to defining chemosensitivity to CPT-induced top1-cleavable complexes and that CPT appears to have two cytotoxic mechanisms, one protectable by aphidicolin, and the other not.

Silencing and selective methylation of the normal topoisomerase I gene in camptothecin-resistant CEM/C2 human leukemia cells.

Camptothecin resistance of the human leukemia CEM/C2 cells is associated with a topoisomerase I (top1) mutation: Asn722Ser (Fujimori, A. et al. Cancer Res. 55:1339-1346; 1995). The corresponding DNA point mutation generates a novel site for the restriction endonuclease DdeI. We found that only the mutated top1 transcript was detectable in CEM/C2 by reverse transcriptase-polymerase chain reaction. Genomic DNA analysis by Southern blotting with DdeI showed that both the mutated and normal top1 genes were present in CEM/C2 cells. The mechanism of normal top1 allele silencing was further investigated. Cytogenetic analysis with a human chromosome 20 specific probe and restriction mapping by Southern blotting showed that both cell lines had a similar copy number of chromosome 20, with the predominant population containing 5-6 copies, and no detectable top1 gene rearrangement. Southern blotting using methylcytosine-sensitive restriction endonuclease (HpaII) indicated differential top1 methylation in CEM/C2 cells. Global cytosine methylation, however, appeared similar in CEM/C2 and wild-type CEM cells. These results indicate that gene-specific DNA methylation can play a role in downregulating top1 gene(s) and in the cellular resistance to camptothecins.

Eukaryotic DNA topoisomerases mediated DNA cleavage induced by a new inhibitor: NSC 665517.

A compound with a novel structure, NSC 665517, was tested in the National Cancer Institute Preclinical Drug Discovery Screen. With the COMPARE algorithm, the pattern of differential cytotoxicity for NSC 665517 most closely resembled those of known topoisomerase II (top2) inhibitors. In vitro data showed that NSC 665517 induced DNA cleavage in the presence of top2 and topoisomerase I (top1) (at a higher concentration). The minimum concentration required to induce top2 cleavage was 0.5 microM. A substantial decrease in top2-induced cleavage by NSC 665517 was seen when the reaction mixtures were shifted to elevated temperature (55 degrees), suggesting that top2-induced cleavage occurs through the mechanism of stabilizing the reversible enzyme/DNA complex and inhibiting religation. The DNA cleavage pattern induced by NSC 665517 with top2 was different than that of other known top2 inhibitors, including etoposide, mitoxantrone, anthracyclines, amsacrine, and ellipticine. top2 cleavage sites induced by NSC 665517 showed strong preference for G located 3' to the top2-mediated DNA cleavage (position +1). NSC 665517 produced limited DNA unwinding at high drug concentration. DNA damage analyzed in KB cells by alkaline elution showed that NSC 665517 induced strand break. Data from the cytotoxicity in KB-V1 overexpressing P-glycoprotein and COMPARE analysis with rhodamine efflux assay indicated that NSC 665517 is a substrate of P-glycoprotein. These results strongly suggest that NSC 665517 is a novel topoisomerase-targeted drug. Preclinical evaluation of NSC 665517 as an antitumor agent is under way.

Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line resistant to camptothecin.

We developed previously a resistant cell line, CEM/C2, from the human leukemia cell line CCRF-CEM by stepwise selection in camptothecin. This cell line is 974-fold more resistant to camptothecin than parental cells. Resistance is only partially explained by 2-fold reductions in topoisomerase I protein and mRNA levels. We further investigated biochemical and molecular features of topoisomerase I in the resistant cell line. Sequence analyses of the top1 cDNA from CEM/C2 identified mutations corresponding to two amino acid substitutions, Met370Thr and Asn722Ser. Asn722Ser is next to the catalytic Tyr723 in a region highly conserved among type I eukaryotic DNA topoisomerases. Recombinant top1 with the corresponding substitution was found to be catalytically active and resistant to camptothecin. These results indicate that camptothecin resistance of CEM/C2 is due to the mutation Asn722Ser and strongly suggest that the asparagine immediately flanking the catalytic tyrosine is important for the camptothecin action.