Rearrangements of the MLL gene are influenced by DNA secondary structure, potentially mediated by topoisomerase II binding.

The location of MLL translocation breakpoints within therapy-related acute myeloid leukemia linked to drugs targeting Topoisomerase II and infant acute leukemia (IAL) are biased toward the intron 11-exon 12 region of MLL, although lacking a comprehensive explanation. To address this, blood samples were taken from breast cancer and lymphoma patients receiving Topoisomerase II inhibitor therapy. Inverse PCR analysis was used to interrogate the exon 12 region of MLL for rearrangements. Eleven of 19 observed translocations showed breakpoint junctions restricted to a single 5 bp location within exon 12. A similarly restricted distribution (11/20 breakpoint junctions) was observed in TK6 cells exposed to either estrogen (linked to IAL) or anti-CD95 antibody. The translocation hotspot was at the 5' edge of a 10-bp tract matched with a perfect palindrome, 101 bp distant. A high stringency Topoisomerase II consensus sequence binding site was noted at the geometric midpoint of the palindromes. Ligation-mediated PCR to screen TK6 cells exposed to anti-CD95 antibody showed 14/37 (38%) of DNA breaks adjacent to the 5' palindrome and 10/37 (27%) at the 3' partner. We propose a model whereby Topoisomerase II facilitates the organization of nuclease-sensitive secondary structures, stabilized by palindrome association, which are prone to rearrangement.

Correlation of GDF5 and connexin 43 mRNA expression during embryonic development.

Growth/differentiation factor 5 (GDF5) regulates connexin expression and enhances embryonic chondrogenesis in a gap junction-dependent manner, suggesting that GDF5 action on developmental skeletogenesis is coordinated with gap junction activities. The results shown here demonstrate concordance between the mRNA expression profiles of GDF5 and the gap junction gene, Cx43, in the mouse embryonic limb, spine, and heart, consistent with coordinated functions for these gene products during developmental organogenesis.

Expression of fibronectin isoforms bearing the alternatively spliced EIIIA, EIIIB, and V segments in corneal alkali burn and keratectomy wound models in the rat.

PURPOSE: To better understand the healing process in the wounded cornea, fibronectin (FN) isoforms bearing the alternatively spliced EIIIA, EIIIB, and V segments (EIIIA+, EIIIB+, and V+ FNs) were evaluated in alkali burn and keratectomy wound models in the rat. METHODS: Alkali burn or keratectomy wounds (both 2 mm) were created, and corneas were harvested at various time points and analyzed by indirect immunofluorescence using antibodies specific for the EIIIA, EIIIB, and V segments as well as for the total pool of FN (total FN). RESULTS: There was minimal staining for any variety of FN in the epithelium or basement membrane zone (BMZ) in normal cornea, but each antibody produced granular staining in the stroma. Bright staining for V+ and total FNs was evident at the denuded stromal surface 1 day following keratectomy. In contrast, staining for EIIIA+ and EIIIB+ FNs was negligible at 24 hours but appeared on the wound surface under the migrating unstained epithelium by the second day. BMZ staining for FN then gradually subsided, such that there was little or no staining by 6 weeks. In contrast, alkali burn wounds exhibited very little BMZ staining throughout the time course. Although there was preferential staining of the anterior aspect of Descemet membrane by anti-EIIIA and anti-EIIIB antibodies under normal conditions, the staining intensity of the anterior and posterior aspects became similar following corneal wounding. CONCLUSION: Deposition of EIIIA+ and EIIIB+ FNs in the BMZ of the keratectomy wound occurs more slowly than deposition of V+ and total FNs. EIIIA+ FN is expressed in a distribution that overlaps with that previously described for the alpha 9 integrin subunit following corneal debridement, suggesting that EIIIA-alpha 9 interactions could occur during corneal wound healing. In contrast, the relative lack of FN deposition in alkali burn wounds suggests that proteolytic degradation of FN may occur; and this, along with impairment of new FN synthesis because of cellular damage, could play a role in the high prevalence of recurrent epithelial erosions in alkali-wounded corneas.

Androgen Receptor regulation of Vitamin D receptor in response of castration-resistant prostate cancer cells to 1α-Hydroxyvitamin D5 - a calcitriol analog.

Calcitriol (1,25(OH)(2)D3) is cytostatic for prostate cancer (CaP), but had limited therapeutic utility due to hypercalcemia-related toxicities, leading to the development of low-calcemic calcitriol analogs. We show that one analog, 1-α-Hydroxyvitamin-D5 (1α(OH)D5), induced apoptosis in castration-sensitive LNCaP prostate cancer cells, but unlike calcitriol, did not increase androgen receptor (AR) transcriptional activity. LNCaP-AI, a castrate-resistant (CRCaP) LNCaP subline, was resistant to 1α(OH)D5 in the presence of androgens; however, androgen withdrawal (AWD), although ineffective by itself, sensitized LNCaP-AI cells to 1α(OH)D5. Investigation of the mechanism revealed that the vitamin D receptor (VDR), which mediates the effects of 1α(OH)D5, is downregulated in LNCaP-AI cells compared to LNCaP in the presence of androgens, whereas AWD restored VDR expression. Since LNCaP-AI cells expressed higher AR compared to LNCaP and AWD decreased AR, this indicated an inverse relationship between VDR and AR. Further, AR stimulation (by increased androgen) suppressed VDR, while AR downregulation (by ARsiRNA) stimulated VDR levels and sensitized LNCaP-AI cells to 1α(OH)D5 similar to AWD. Another cell line, pRNS-1-1, although isolated from a normal prostate, had lost AR expression in culture and adapted to androgen-independent growth. These cells expressed the VDR and were sensitive to 1α(OH)D5, but restoration of AR expression suppressed VDR levels and induced resistance to 1α(OH)D5 treatment. Taken together, these results demonstrate negative regulation of VDR by AR in CRCaP cells. This effect is likely mediated by prohibitin (PHB), which was inhibited by AR transcriptional activity and stimulated VDR in CRCaP, but not castrate-sensitive cells. Therefore, in castration sensitive cells, although the AR negatively regulates PHB, this does not affect VDR expression, whereas in CRCaP cells, negative regulation of PHB by the AR results in concomitant negative regulation of the VDR by the AR. These data demonstrate a novel mechanism by which 1α(OH)D5 prolong the effectiveness of AWD in CaP cells.

Estrogen treatment induces MLL aberrations in human lymphoblastoid cells.

Epidemiological data indicates increased risk of infant acute leukemia involving MLL gene aberrations with use of oral contraceptives. To determine whether estrogens might be implicated, we examined the effect of estradiol (E2) or 4-OH-E2 in an in vitro model of translocation susceptibility. Genomic DNA from the TK6 human lymphoblastoid cell line was screened by ligation mediated PCR and inverse PCR at a rearrangement hot spot within the MLL breakpoint cluster region to detect DNA aberrations. An increase in DNA double strand breaks was observed within this region after exposure to either E2 or 4-OH-E2. An increase in the frequency of MLL translocations was only found after exposure to E2. Induction of cleavage due to increased activation of apoptotic nucleases was excluded by pre-treatment with the pan-caspase inhibitor, zVAD.fmk. We conclude that concentrations of E2 and 4-OH-E2 that may occur during pregnancy, or during use of oral contraceptives, can cause aberrations of the MLL gene and could thus be a factor in the early events of leukemogenesis occurring in utero.

Electrophoretic characterization of species of fibronectin bearing sequences from the N-terminal heparin-binding domain in synovial fluid samples from patients with osteoarthritis and rheumatoid arthritis.

Fragments of fibronectin (FN) corresponding to the N-terminal heparin-binding domain have been observed to promote catabolic chondrocytic gene expression and chondrolysis. We therefore characterized FN species that include sequences from this domain in samples of arthritic synovial fluid using one-and two-dimensional (1D and 2D) Western blot analysis. We detected similar assortments of species, ranging from ~47 to greater than 200 kDa, in samples obtained from patients with osteoarthritis (n = 9) versus rheumatoid arthritis (n = 10). One of the predominant forms, with an apparent molecular weight of ~170 kDa, typically resolved in 2D electrophoresis into a cluster of subspecies. These exhibited reduced binding to gelatin in comparison with a more prevalent species of ~200+ kDa and were also recognized by a monoclonal antibody to the central cell-binding domain (CBD). When considered together with our previous analyses of synovial fluid FN species containing the alternatively spliced EIIIA segment, these observations indicate that the ~170-kDa species includes sequences from four FN domains that have previously, in isolation, been observed to promote catabolic responses by chondrocytes in vitro: the N-terminal heparin-binding domain, the gelatin-binding domain, the central CBD, and the EIIIA segment. The ~170-kDa N-terminal species of FN may therefore be both a participant in joint destructive processes and a biomarker with which to gauge activity of the arthritic process.

Plasma levels of fibronectin bearing the alternatively spliced EIIIB segment are increased after major trauma.

Using Western-blot analysis and enzyme-linked immunosorbent assay (ELISA) of N-deglycosylated samples, we have observed that plasma levels of fibronectin (FN) bearing the alternatively spliced EIIIB segment (EIIIB(+) FN) increase in patients after admission to the intensive-care unit (ICU) for acute major trauma. Although not increased at the first sampling ("0 hour"), taken within 24 hours of ICU admission, levels measured 24, 48, and 72 hours later were significantly increased compared with levels obtained in healthy controls. Furthermore, average concentrations at these latter time points were significantly increased relative to the 0-hour sampling. EIIIB(+) FN levels then decreased in plasma samples taken 1 month after hospital discharge, such that no significant difference was found between ELISA-measured values at this time and 0 hour or control values. On the basis of comparisons with previous measurements in these samples, it is apparent that after acute major trauma, circulating levels of soluble EIIIB(+) FN exhibit temporal changes that are qualitatively similar to those encountered for FN isoforms bearing the alternatively spliced EIIIA segment (EIIIA(+) FN), yet different from those observed for the total pool of circulating FN. This is the first report of measurement of FN in clinical plasma samples with antibodies specific for the highly conserved EIIIB segment. Like EIIIA(+) forms of FN, EIIIB(+) FNs are recognized as soluble bloodborne markers for vascular tissue injury.