The SET and transposase domain protein Metnase enhances chromosome decatenation: regulation by automethylation.

Metnase is a human SET and transposase domain protein that methylates histone H3 and promotes DNA double-strand break repair. We now show that Metnase physically interacts and co-localizes with Topoisomerase IIalpha (Topo IIalpha), the key chromosome decatenating enzyme. Metnase promotes progression through decatenation and increases resistance to the Topo IIalpha inhibitors ICRF-193 and VP-16. Purified Metnase greatly enhanced Topo IIalpha decatenation of kinetoplast DNA to relaxed circular forms. Nuclear extracts containing Metnase decatenated kDNA more rapidly than those without Metnase, and neutralizing anti-sera against Metnase reversed that enhancement of decatenation. Metnase automethylates at K485, and the presence of a methyl donor blocked the enhancement of Topo IIalpha decatenation by Metnase, implying an internal regulatory inhibition. Thus, Metnase enhances Topo IIalpha decatenation, and this activity is repressed by automethylation. These results suggest that cancer cells could subvert Metnase to mediate clinically relevant resistance to Topo IIalpha inhibitors.

Phase II studies of antiangiogenic four drug regimens for the treatment of advanced renal cell carcinoma: FUNIL-retinoid and the FUNIL-thalidomide protocols.

BACKGROUND AND PURPOSE: The objective of these studies was to determine the activity of two alternative 4- drug combinations using cis-retinoic acid or thalidomide administered with a previously developed combination of 5 fluorouracil, interferon-alpha, and interleukin 2 (FUNIL), for patients with metastatic renal cell carcinoma (RRC). METHODS: Patients enrolled in these studies had progressive measurable metastatic renal cell cancer and signed an informed consent. Treatments included continuous infusions of 5-fluorouracil, interferon-alpha, 6 MIU/m2 given subcutaneous on days 1, 3, and 5 every week, interleukin-2 6 MIU/m2/day given by continuous infusion days 2 to 5 every week, and either cis-retinoic acid at a dose of 1 mg/kg/day orally in two divided doses or thalidomide given at an initial dose of 200 mg per day. Each cycle consisted of 6 or 4 weeks of the combinations, respectively, followed by a 2-week rest. Patients were evaluated for response prior to each successive cycle. A 2-step mini-max statistical design was used. RESULTS: In the cis-retinoid study, 20 patients were enrolled. One patient was ineligible. There were 1 complete and 2 partial responses (one confirmed and one unconfirmed) (15.8%), 1 stable disease, and 15 disease progression. In the thalidomide combination study, 20 patients were enrolled, but only 19 are assessable. One patient progressed early and was never treated. There were 2 partial responses (10.5%), 4 stable disease, and 13 progressive disease. CONCLUSION: Neither the FUNIL-cis-retinoid nor the FUNIL-thalidomide regimens met their primary objective first step endpoint of 3 confirmed responses. Both regimens had significant adverse effects and neither is considered promising for further study.

The chemokine CXCL14 (BRAK) stimulates activated NK cell migration: implications for the downregulation of CXCL14 in malignancy.

OBJECTIVE: The primary function of chemokines is the regulation of leukocyte trafficking by stimulating directional chemotaxis. The chemokine CXCL14 (BRAK) is highly expressed in all normal tissues, but is not expressed in most malignant tissues. The chemotactic activity of CXCL14 has been difficult to characterize. Recently it was reported that CXCL14 is a chemoattractant for activated monocytes and immature dendritic cells. Given that CXCL14 is downregulated upon transition to malignancy, we sought to characterize whether CXCL14 might play a role in NK cell chemotaxis. METHODS: Human natural killer (NK) cells were isolated from buffy coats obtained from normal volunteers and were activated with lymphocyte conditioned media, IL-2, and ionomycin. Standard transwell chemotaxis assays, proliferation assays, and chromium release cell cytotoxicity assays were performed. RESULTS: CXCL14 was found to stimulate migration of activated human NK cells in transwell chemotaxis assays by 1.4-fold. Similarly, it increased migration of an IL-2-dependent natural killer leukemia (NKL) cell line by 1.9-fold. Antisera against CXCL14 or pertussis toxin blocked this chemotactic effect. However, CXCL14 did not affect the proliferation or cytotoxic activity of normal human NK cells. CXCL14 also stimulated the chemotaxis of immature monocyte-derived dendritic cells. CONCLUSIONS: CXCL14 may play a role in the trafficking of NK cells to sites of inflammation or malignancy. In addition, the downregulation of the expression of CXCL14 might be an important step in successful oncogenesis to prevent NK immune surveillance of the malignancy.

The SET domain protein Metnase mediates foreign DNA integration and links integration to nonhomologous end-joining repair.

The molecular mechanism by which foreign DNA integrates into the human genome is poorly understood yet critical to many disease processes, including retroviral infection and carcinogenesis, and to gene therapy. We hypothesized that the mechanism of genomic integration may be similar to transposition in lower organisms. We identified a protein, termed Metnase, that has a SET domain and a transposase/nuclease domain. Metnase methylates histone H3 lysines 4 and 36, which are associated with open chromatin. Metnase increases resistance to ionizing radiation and increases nonhomologous end-joining repair of DNA doublestrand breaks. Most significantly, Metnase promotes integration of exogenous DNA into the genomes of host cells. Therefore, Metnase is a nonhomologous end-joining repair protein that regulates genomic integration of exogenous DNA and establishes a relationship among histone modification, DNA repair, and integration. The data suggest a model wherein Metnase promotes integration of exogenous DNA by opening chromatin and facilitating joining of DNA ends. This study demonstrates that eukaryotic transposase domains can have important cell functions beyond transposition of genetic elements.