full-length human parathyroid car cdna  (NPS corporation)

Journal: International journal of cancer

Article Title: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma.

doi: 10.1002/ijc.22749

Figure Lengend Snippet: FIGURE 1 – PTHR1 and RANKL mRNA level in osteosarcoma xen- ografts and cultured cell lines. The levels of PTHR1 mRNA and RANKL mRNA were measured by qRT-PCR in 4 osteosarcoma xeno- grafts, 5 osteosarcoma patient sample derived cell lines and 4 osteo- sarcoma standard cell lines. The relative quantitiy of mRNA was calculated based on the methodology: 22DCt as described previously25 (DCT 5 CT gene of interest- CT endogenous control). PTHR1 and RANKL mRNA are higher in osteosarcoma xenografts than those in patient samples derived osteosarcoma cell lines respectively (p < 0.05, Wilcoxon rank sum test), and those in standard cell lines respec- tively (p < 0.05). Each dot represents 1 data point and median levels of mRNA in each group are denoted by black lines.

Article Snippet: Full-length human PTHR1 cDNA was purchased from Origene (Rockville, MD) and subcloned into the Not I site of pcDNA3.1 (Invitrogen, Bethesda, MD).

Techniques: Cell Culture, Quantitative RT-PCR, Derivative Assay, Control

Journal: International journal of cancer

Article Title: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma.

doi: 10.1002/ijc.22749

Figure Lengend Snippet: FIGURE 2 – PTHR1 mRNA level is higher in metastatic/relapsed osteosarcoma samples. PTHR1 mRNA was measured in 55 patient specimens using real-time qRT-PCR. Samples from metastatic/ relapsed sites (n 5 25) had significant higher levels of PTHR1 mRNA than those from primary sites (n 5 30) (p < 0.01, Wilcoxon rank sum test). The black lines indicating the median value of PTHR1 mRNA in each group, the plot box includes samples with expression level rang- ing between 25th and 75th quartiles in each group. Clouds outside the whiskers represent statistical outliers.

Article Snippet: Full-length human PTHR1 cDNA was purchased from Origene (Rockville, MD) and subcloned into the Not I site of pcDNA3.1 (Invitrogen, Bethesda, MD).

Techniques: Quantitative RT-PCR, Expressing

Journal: International journal of cancer

Article Title: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma.

doi: 10.1002/ijc.22749

Figure Lengend Snippet: FIGURE 3 – PTHR1 over-expressing osteo- sarcoma model was established in vitro. Panel A and B: PTHR1 (Panel A) and PTHrP (Panel B) mRNA levels were measured in HOS-PTHR1-C5 and HOS-PTHR1-C7 as compared with empty vector-only HOS cells. Relative folds difference of mRNA levels measured in PTHR1 transfected cells over the vector-only transfected control are shown respectively. Experiments were performed 3 times; mean and standard deviation are shown. Empty bar, HOS-Vector-only; gray bar, HOS-PTHR1-C5; and black bar, HOS- PTHR1-C7. The protein expressions were confirmed by Western Blots. Panel C: Cyclic AMP was measured in at basal level (after se- rum starvation over night), and after incuba- tion with 1028 M PTHrP (1–34) or 5 lM H89, for 30 min and were normalized by pro- tein amount in each well. At basal level, a significantly higher concentration of cAMP was detected in both PTHR1 transfected HOS sub-lines. After PTHrP incubation, a signifi- cantly higher level of cAMP was detected in HOS-PTHR1-C7 cells as compared with the control, and a trend of that was detected in HOS-PTHR1-C5 cells. Cyclic AMP produc- tion was nearly completely inhibited by adenylyl cyclase (AC) inhibitor H89. Experi- ments were performed in duplicate; mean and standard deviation of each sample are indi- cated. * indicates p < 0.05, and ** indicates p < 0.01 as compared with the vector-only transfected HOS cells in each group, respec- tively.

Article Snippet: Full-length human PTHR1 cDNA was purchased from Origene (Rockville, MD) and subcloned into the Not I site of pcDNA3.1 (Invitrogen, Bethesda, MD).

Techniques: Expressing, In Vitro, Plasmid Preparation, Transfection, Control, Standard Deviation, Western Blot, Concentration Assay, Incubation

Journal: International journal of cancer

Article Title: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma.

doi: 10.1002/ijc.22749

Figure Lengend Snippet: FIGURE 4 – Cell proliferation Assay. Panel A: 4,000 cells/200 ll were seeded in 96-well plate in media containing 5% dialyzed fetal bovine serum in the presence or absence of 1028 M of PTHrP (1–34), respectively. Cell number was measured at day 0, 1, 2 and day 3 as indicated. Mean value of relative cell number and standard deviation derived from 2 independent experiments are plotted as function of time point. Panel B: Cell proliferation assay with PKA or PKC inhibi- tors. Cell were treated as described in Figure 4a in the presence of PKA inhibitor H89 at a concentration of 5 lM, or PKC inhibitor che- lethyrine chloride (ChCL) at a concentration of 1 lM. Cell number was measured at day 0, 1, 2 and day 3, and was presented as percent- age relative to respective nontreated ones. Cell proliferation was inhibited by PKC inhibitor chelerythrine chloride (ChCl) preferen- tially in PTHR1 transfected HOS cells, but not by the PKA inhibitor H89. Two independent experiments were performed. Inset, activation of PKCs was detected by Western blot probed with indicated p-PKC (pan) Ser660 antibody. Total PKC was used as a loading control.

Article Snippet: Full-length human PTHR1 cDNA was purchased from Origene (Rockville, MD) and subcloned into the Not I site of pcDNA3.1 (Invitrogen, Bethesda, MD).

Techniques: Proliferation Assay, Standard Deviation, Derivative Assay, Concentration Assay, Transfection, Activation Assay, Western Blot, Control

Journal: International journal of cancer

Article Title: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma.

doi: 10.1002/ijc.22749

Figure Lengend Snippet: FIGURE 5 – Panel A: Migration Assay Wound healing assay was used to assess the motility of transfected cells (data not shown), and was quantitatively validated by Boyden chamber migration assay. Cells were treated as described in Methods. Cells migrated into lower chamber were stained with 0.2% hematoxylin. Cell number was counted for 5 highpower fields for each chamber and presented as migrated cells number/field. Triplicate experiments were performed and data were presented as Mean 6 STDEV. * indicates p < 0.05, and *** indicates p < 0.001 as compared with the vector-only trans- fected HOS cells in each group, respectively. Panel B: Matrigel Inva- sion Assay. Invasion assays were performed using 24-well invasion chamber system. After 36 hr, Nonmigratory cells in the upper cham- ber were removed. Invaded cells on the lower surface stained with 0.2% hematoxylin are shown for PTHR1 transfected HOS sub-lines and the empty vector-only transfected HOS cell line, respectively. Invaded cells were counted under a light microscope. Cells placed in noncoated Boyden chamber for 18 hr were used to normalize cell number as described.27 Average invasion indices and standard devia- tion from 3 independent experiments were shown. ** indicates p < 0.005, and *** indicates p < 0.001 as compared with the vector-only transfected HOS cells in each group, respectively.

Article Snippet: Full-length human PTHR1 cDNA was purchased from Origene (Rockville, MD) and subcloned into the Not I site of pcDNA3.1 (Invitrogen, Bethesda, MD).

Techniques: Migration, Wound Healing Assay, Transfection, Staining, Plasmid Preparation, Light Microscopy

Journal: International journal of cancer

Article Title: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma.

doi: 10.1002/ijc.22749

Figure Lengend Snippet: FIGURE 6 – TGF-b1 and CTGF mRNA measurements. PTHR1 transfected HOS sub-lines showed significantly higher levels of TGF- b1 and CTGF mRNA than empty vector-only transfected ones as measured by qRT–PCR. Average of relative mRNA quantity and standard deviation from 3 independent experiments are shown. ** indicates p < 0.005, and *** indicates p < 0.001 as compared with the vector-only transfected HOS cells in each group, respectively.

Article Snippet: Full-length human PTHR1 cDNA was purchased from Origene (Rockville, MD) and subcloned into the Not I site of pcDNA3.1 (Invitrogen, Bethesda, MD).

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation

Journal: International journal of cancer

Article Title: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma.

doi: 10.1002/ijc.22749

Figure Lengend Snippet: FIGURE 8 – Osteoblastic differ- entiation. After 2 weeks of induc- tion, less alkaline phosphatase (ALP staining) and less bone min- eralization nodules (Alizarin Red S staining) were formed in both PTHR1 transfected HOS sub-lines as compared with empty vector- only transfected HOS cells.

Article Snippet: Full-length human PTHR1 cDNA was purchased from Origene (Rockville, MD) and subcloned into the Not I site of pcDNA3.1 (Invitrogen, Bethesda, MD).

Techniques: Staining, Transfection, Plasmid Preparation

Journal: International journal of cancer

Article Title: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma.

doi: 10.1002/ijc.22749

Figure Lengend Snippet: FIGURE 10 – Schematic model of PTHR1 mediated signaling in os- teosarcoma. PTHrP (P) activates PTHR1 and the coupled heterotri- meric G proteins (a and bg subunit) relay the signals to 2 classic down- stream pathways: (i) Adenylyl Cy- clase (AC)—cyclic AMP- Protein Kinase A (PKA); (ii) Phospholipase C (PLC)—Protein Kinase C (PKC). Increased cell proliferation caused by PTHR1 ectopic expression ap- peared to be associated with PKC activation in this study. PTHrP/ PTHR1 signaling induces TGF-b1 up-regulation as suggested by the current study and previous study,46 which in turn, activates its cognate receptors and leads to Smads-medi- ated PTHrP up-regulation,21,47 therefore forming a positive feed- back loop. TGF-b1 and connective tissue growth factor (CTGF) are involved in extrucellular matrix (ECM) production and remodel- ing.22 In addition, PTHrP/PTHR1 signaling increases osteoclastogene- sis through the RANKL/OPG sys- tem. Osteoclasts mediate bone ma- trix destruction and bone resorption to facilitate the expansive tumor growth in bone.

Article Snippet: Full-length human PTHR1 cDNA was purchased from Origene (Rockville, MD) and subcloned into the Not I site of pcDNA3.1 (Invitrogen, Bethesda, MD).

Techniques: Expressing, Activation Assay

Journal: International journal of cancer

Article Title: Over-expression of parathyroid hormone Type 1 receptor confers an aggressive phenotype in osteosarcoma.

doi: 10.1002/ijc.22749

Figure Lengend Snippet: FIGURE 9 – Osteoclastogenesis. Osteo- clasts formed from co-culture of bone mar- row-derived monocytes with transfected HOS cells for 5 days, were TRAP stained (arrows) and counted under a light microscope. A sig- nificant higher number of osteoclasts were formed from co-culture with PTHR1 trans- fected HOS sub-lines as compared with that with empty vector-only transfected HOS cells. Average numbers of osteoclasts and standard deviation from 3 independent ex- periments are shown. * indicates p < 0.05, and ** indicates p < 0.005 as compared with the vector-only transfected HOS cells, respec- tively.

Article Snippet: Full-length human PTHR1 cDNA was purchased from Origene (Rockville, MD) and subcloned into the Not I site of pcDNA3.1 (Invitrogen, Bethesda, MD).

Techniques: Co-Culture Assay, Derivative Assay, Transfection, Staining, Light Microscopy, Plasmid Preparation, Standard Deviation