Characterization of ABCG2 gene amplification manifesting as extrachromosomal DNA in mitoxantrone-selected SF295 human glioblastoma cells

The human ABCG2 gene, located on chromosome 4, encodes an ATP-binding cassette half-transporter that has been shown to confer resistance to chemotherapeutic agents. Relatively little is known about the mechanisms controlling expression of ABCG2. In previous studies, we had shown that overexpression of ABCG2 can result from rearrangement or gene amplification involving chromosome 4. To better characterize the mechanisms of ABCG2 overexpression, SF295 glioblastoma cells were exposed to increasing amounts of mitoxantrone to generate the SF295 MX50, MX100, MX250, and MX500 sublines, maintained in mitoxantrone concentrations ranging from 50 to 500 nmol/L. Northern blot analysis confirmed overexpression of ABCG2 mRNA, and immunoblot analysis demonstrated increased protein expression in the selected cell lines. Efflux of BODIPY-prazosin confirmed a functional protein. ABCG2 gene amplification was observed in all resistant sublines, as determined by Southern blot analysis. Fluorescence in situ hybridization (FISH) revealed amplification of ABCG2 via double minute chromosomes (dmins) detected in metaphase chromosome spreads in the SF295 MX50 and MX100 sublines. At higher levels of drug selection, in the MX250 and MX500 sublines, fewer dmins were observed but homogeneously staining regions (hsr) were visible with FISH analysis, revealing reintegration of the ABCG2 gene into multiple chromosomes. Spectral karyotyping (SKY) demonstrated multiple clonal and nonclonal rearrangements of chromosome 4, including hsrs. These results suggest that amplification of ABCG2 occurred initially in the form of dmins, followed by chromosomal reintegration of the amplicon at multiple sites. This occurred with increasing drug-selection pressure, generating a more stable genotype.     

Side population analysis using a violet-excited cell-permeable DNA binding dye

Hoechst 33342 side population (SP) analysis is a common method for identifying stem cells in mammalian hematopoietic and nonhematopoietic tissues. Although widely employed for stem cell analysis, this method requires an ultraviolet (UV) laser to excite Hoechst 33342. Flow cytometers equipped with UV sources are not common because of the cost of both the laser and optics that can transmit light UV light. Violet laser sources are inexpensive and are now common fixtures on flow cytometers, but have been previously shown to provide insufficient Hoechst dye excitation for consistent resolution of SP cells. One solution to this problem is to identify additional fluorescent substrates with the same pump specificity as Hoechst 33342, but with better violet excitation characteristics. DyeCycle Violet reagent has emission characteristics similar to those of Hoechst 33342, but with a longer wavelength excitation maxima (369 nm). When this dye is loaded into hematopoietic cells, a sharply resolved side population was also observed, similar in appearance to that seen with Hoechst 33342. Unlike Hoechst SP, DCV SP was similar in appearance with both violet and UV excitation. DCV SP could be inhibited fumitremorgin C, and showed the same membrane pump specificity as Hoechst 33342. Simultaneous immunophenotyping with stem cell markers in mouse bone marrow demonstrated that DCV SP was restricted to the stem cell lineage(-) Sca-1(+) c-kit(+) cells population, as is Hoechst SP. Pending confirmation by functional analysis of DCV SP cells, these results suggest that DCV efflux identified approximately the same stem cell population as did Hoechst 33342 efflux. Substituting DCV for Hoechst 33342 in the SP technique may, therefore, allow side population analysis on flow cytometers with violet lasers.     

RANKL Inhibition in Fibrous Dysplasia of Bone: A Preclinical Study in a Mouse Model of the Human Disease

Fibrous dysplasia of bone/McCune-Albright syndrome (Polyostotic FD/MAS; OMIM#174800) is a crippling skeletal disease caused by gain-of-function mutations of G_sα. Enhanced bone resorption is a recurrent histological feature of FD and a major cause of fragility of affected bones. Previous work suggests that increased bone resorption in FD is driven by RANKL and some studies have shown that the anti-RANKL monoclonal antibody, denosumab, reduces bone turnover and bone pain in FD patients. However, the effect of RANKL inhibition on the histopathology of FD and its impact on the natural history of the disease remain to be assessed. In this study, we treated the EF1α-G_sα^R201C mice, which develop an FD-like phenotype, with an anti-mouse RANKL monoclonal antibody. We found that the treatment induced marked radiographic and microscopic changes at affected skeletal sites in 2-month-old mice. The involved skeletal segments became sclerotic due to the deposition of new, highly mineralized bone within developing FD lesions and showed a higher mechanical resistance compared to affected segments from untreated transgenic mice. Similar changes were also detected in older mice with a full-blown skeletal phenotype. The administration of anti-mouse RANKL antibody arrested the growth of established lesions and, in young mice, prevented the appearance of new ones. However, after drug withdrawal, the newly formed bone was remodelled into FD tissue and the disease progression resumed in young mice. Taken together, our results show that the anti-RANKL antibody significantly affected the bone pathology and natural history of FD in the mouse. Pending further work on the prevention and management of relapse after treatment discontinuation, our preclinical study suggests that RANKL inhibition may be an effective therapeutic option for FD patients. © 2019 American Society for Bone and Mineral Research.     

不同手术方法治疗不稳定Hangman骨折的疗效观察

目的探讨不同手术方法治疗不稳定Hangman骨折的临床疗效。方法 14例不稳定Hangman骨折患者根据Levine-Edwards分型:Ⅱ型7例,ⅡA型5例,Ⅲ型2例。4例行后路C2,3椎弓根螺钉内固定术,7例行前路C2,3间盘摘除减压植骨融合内固定术,3例行前后路联合手术。结果患者均获随访,时间3~24个月。1例患者术后3个月单侧椎弓根处可见模糊骨折线,13例患者均达到骨性愈合。术中及术后未出现椎动脉、脊髓损伤及脑脊液漏等并发症,未发生钢板螺钉断裂内固定失效等现象。结论对于不稳定Hangman骨折无论是采取前路或者后路手术均可使骨折达到骨性愈合,对严重骨折伴脱位Hangman骨折可以采取前后路联合的手术方式。     

Phenotypic effects of biglycan deficiency are linked to collagen fibril abnormalities, are synergized by decorin deficiency, and mimic Ehlers-Danlos-like changes in bone and other connective tissues.

Decorin (dcn) and biglycan (bgn), two members of the family of small leucine-rich proteoglycans (SLRPs), are the predominant proteoglycans expressed in skin and bone, respectively. Targeted disruption of the dcn gene results in skin laxity and fragility, whereas disruption of the bgn gene results in reduced skeletal growth and bone mass leading to generalized osteopenia, particularly in older animals. Here, we report that bgn deficiency leads to structural abnormality in collagen fibrils in bone, dermis, and tendon, and to a "subclinical" cutaneous phenotype with thinning of the dermis but without overt skin fragility. A comparative ultrastructural study of different tissues from bgn- and dcn-deficient mice revealed that bgn and dcn deficiency have similar effects on collagen fibril structure in the dermis but not in bone. Ultrastructural and phenotypic analysis of newly generated bgn/dcn double-knockout (KO) mice revealed that the effects of dcn and bgn deficiency are additive in the dermis and synergistic in bone. Severe skin fragility and marked osteopenia characterize the phenotype of double-KO animals in which progeroid changes are observed also in the skin. Ultrastructural analysis of bone collagen fibrils in bone of double-KO mice reveals a complete loss of the basic fibril geometry with the emergence of marked "serrated fibril" morphology. The phenotype of the double-KO animal mimics directly the rare progeroid variant of human Ehlers-Danlos syndrome (EDS), in which skin fragility, progeroid changes in the skin (reduced hypodermis), and osteopenia concur as a result of impaired glycosaminoglycan (GAG) linking to bgn and dcn core proteins. Our data show that changes in collagen fibril morphology reminiscent of those occurring in the varied spectrum of human EDS are induced by both bgn deficiency and den deficiency in mice. The effects of an individual SLRP deficiency are tissue specific, and the expression of a gross phenotype depends on multiple variables including level of expression of individual SLRPs in different tissues and synergisms between different SLRPs (and likely other macromolecules) in determining matrix structure and functional properties.