Bolander, Mark E.

Abstract Purpose: Although metastasis is the primary determinant of poor survival of patients with osteogenic sarcoma, some patients live much longer than others, indicating metastatic heterogeneity underlying survival outcome. The purpose of the investigation was to identify genes underlying survival outcome of patients with osteogenic sarcoma metastasis. Experimental Design: We have used microarray to first compare mRNA expression between normal bone and osteogenic sarcoma specimens, identified genes overexpressed in osteogenic sarcoma, and compared expression of the selected gene between a poorly metastatic (SAOS) and two highly metastatic cell lines (LM8 and 143B). Finally, expression of the selected gene was assessed by immunostaining of osteogenic sarcoma samples with known survival outcome. Results: Microarray analysis revealed 5.3-fold more expression of WT1 mRNA in osteogenic sarcoma compared with normal bone and >2-fold overexpression in 143B and LM8 cells compared with SAOS. Furthermore, WT1 mRNA was absent in normal bone (10 of 10) by reverse transcription-PCR but present in osteogenic sarcoma–derived cell lines (5 of 8). One hundred percent (42 of 42) of low-grade osteogenic sarcoma specimens expressed no WT1 as determined by immunostaining; however, 24% (12 of 49) of the high-grade specimens showed intense staining. Mean survival of patients with high-grade metastatic osteogenic sarcoma but low WT1 staining (27 of 37) was 96.5 ± 129.3 months, whereas mean survival of patients with high-grade metastatic osteogenic sarcoma having intense staining (10 of 37) was 18.3 ± 12.3 months (P > 0.0143). All splice variants of WT1 mRNA, including a hitherto unknown variant (lacking exons 4 and 5), were found to be expressed in osteogenic sarcoma. Conclusion: WT1 seems to be associated with very poor survival of patients with osteogenic sarcoma metastasis.

Abstract Increasing evidence suggests a potential role for activin in bone formation. However, the cognate receptors through which activins function with respect to skeletal tissues have not yet been identified. Identification and regulation of expression of these receptors are necessary prerequisites to understanding the role of activins in bone metabolism. We detected mRNAs for three activin receptors, type I (ActRI), type II (ActRII), and type IIB (ActRIIB), in multiple skeletal tissues in rat, including tibia and costochondral growth plate, and also in cultured osteoblasts. To gain information about the relationship between receptor expression and different skeletal cell functions, we evaluated expression of the three receptors in a semiquantitative manner during the early stages of fracture healing, a model for rapid bone formation. Relatively high levels of ActRI and ActRII expression were detected in the callus at 7, 10, and 14 days after fracture, times that correlate with the interval of rapid intramembranous bone formation and the initiation of endochondral bone formation. Expression of the ActRIIB in the fracture callus was strikingly lower than either ActRI or ActRII. Immunostaining of the fracture callus and the newborn rat femur with an anti-ActRII antibody localized the receptor to osteoblasts at regions of membranous and endochondral bone formation. No staining of osteoblasts in fracture callus or bone was seen with an anti-ActRIIB antibody. These results provide strong evidence of the identification of the principal receptors through which activins could function in the skeletal system and further shed light on activin's mechanism of action in bone formation.

Abstract Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene. Introduction: Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors. Materials and Methods: To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction. Results: The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14–21 days) with insulin. Conclusion: These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA.

Abstract We examined the time course effects of continuous PTH on cortical bone and mechanical properties. PTH increased cortical bone turnover and induced intracortical porosity with no deleterious effect on bone strength. Withdrawal of PTH increased maximum torque to failure and stiffness with no change in energy absorbed. Introduction: The skeletal response of cortical bone to parathyroid hormone (PTH) is complex and species dependent. Intermittent administration of PTH to rats increases periosteal and endocortical bone formation but has no known effects on intracortical bone turnover. The effects of continuous PTH on cortical bone are not clearly established. Materials and Methods: Eighty-four 6-month-old female Sprague-Dawley rats were divided into three control, six PTH, and two PTH withdrawal (WD) groups. They were subcutaneously implanted with osmotic pumps loaded with vehicle or 40 μg/kg BW/day human PTH(1-34) for 1, 3, 5, 7, 14, and 28 days. After 7 days, PTH was withdrawn from two groups of animals for 7 (7d-PTH/7d-WD) and 21 days (7d-PTH/21d-WD). Histomorphometry was performed on periosteal and endocortical surfaces of the tibial diaphysis in all groups. μCT of tibias and mechanical testing by torsion of femora were performed on 28d-PTH and 7d-PTH/21d-WD animals. Results and Conclusions: Continuous PTH increased periosteal and endocortical bone formation, endocortical osteoclast perimeter, and cortical porosity in a time-dependent manner, but did not change the mechanical properties of the femur, possibly because of addition of new bone onto periosteal and endocortical surfaces. Additionally, withdrawal of PTH restored normal cortical porosity and increased maximum torque to failure and stiffness. We conclude that continuous administration of PTH increased cortical porosity in rats without having a detrimental effect on bone mechanical properties.

Many methods are available for transferring foreign genes into mammalian cells (transfection). The advantages of ultrasound-mediated transfection are that it can be controlled in both space, by focusing, and time, by exposure. It was tested whether ultrasound-mediated gene transfection in cultured immortalized human chondrocytes would be enhanced using artificial cavitation nuclei in the form of Albunex®. Here 1.0 MHz ultrasound transmitted through the bottoms of six-well culture plates containing media with green fluorescent protein reporter gene plasmids at a concentration of 40 μg/ml and Albunex® at 50×106 bubbles/ml produced a peak transfection efficiency of about 50% of the living cells when exposure was 4×105 P spatial average peak pressure (SAPP) for 20 mins. Using these parameters, transfection efficiency increased linearly with ultrasound exposure pressure with a transfection threshold observed at a SAPP of 105 P. Adding fresh Albunex® at 50×106 bubbles/ml prior to sequential one second 3.2 or 4.0×105 P exposures increased transfection efficiency by 15% for each of three consecutive exposures. Efficient in vitro and perhaps in vivo transfection appears possible with ultrasound providing both temporal and spatial control over the transfection process.