Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation

<p>Abstract</p> <p>Background</p> <p>Adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1); however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated.</p> <p>Results</p> <p>We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R) in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1α (MIP-1α), a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was enhanced due to activation of lymphocytes by HTLV-1 infection.</p> <p>Conclusion</p> <p>These data demonstrate that PTHrP and its receptor are up-regulated specifically during immortalization of T-lymphocytes by HTLV-1 infection and may facilitate the transformation process.</p

Incomplete but Infectious Vaccinia Virions Are Produced in the Absence of Oncolysis in Feline SCCF1 Cells

Vaccinia virus is a large, enveloped virus of the poxvirus family. It has broad tropism and typically virus replication culminates in accumulation and lytic release of intracellular mature virus (IMV), the most abundant form of infectious virus, as well as release by budding of extracellular enveloped virus (EEV). Vaccinia viruses have been modified to replicate selectively in cancer cells and clinically tested as oncolytic agents. During preclinical screening of relevant cancer targets for a recombinant Western Reserve strain deleted for both copies of the thymidine kinase and vaccinia growth factor genes, we noticed that confluent monolayers of SCCF1 cat squamous carcinoma cells were not destroyed even after prolonged infection. Interestingly, although SCCF1 cells were not killed, they continuously secreted virus into the cell culture supernatant. To investigate this finding further, we performed detailed studies by electron microscopy. Both intracellular and secreted virions showed morphological abnormalities on ultrastructural inspection, suggesting compromised maturation and morphogenesis of vaccinia virus in SCCF1 cells. Our data suggest that SCCF1 cells produce a morphologically abnormal virus which is nevertheless infective, providing new information on the virus-host cell interactions and intracellular biology of vaccinia virus.Peer reviewe

Canine Prostate Cancer Cell Line (Probasco) Produces Osteoblastic Metastases In Vivo

In 2012, over 240,000 men were diagnosed with prostate cancer and over 28,000 died from the disease. Animal models of prostate cancer are vital to understanding its pathogenesis and developing therapeutics. Canine models in particular are useful due to their similarities to late-stage, castration-resistant human disease with osteoblastic bone metastases. This study established and characterized a novel canine prostate cancer cell line that will contribute to the understanding of prostate cancer pathogenesis

The ARF Tumor Suppressor Regulates Bone Remodeling and Osteosarcoma Development in Mice

The ARF tumor suppressor regulates p53 as well as basic developmental processes independent of p53, including osteoclast activation, by controlling ribosomal biogenesis. Here we provide evidence that ARF is a master regulator of bone remodeling and osteosarcoma (OS) development in mice. Arf-/- mice displayed increased osteoblast (OB) and osteoclast (OC) activity with a significant net increase in trabecular bone volume. The long bones of Arf-/- mice had increased expression of OB genes while Arf-/- OB showed enhanced differentiation in vitro. Mice transgenic for the Tax oncogene develop lymphocytic tumors with associated osteolytic lesions, while Tax+Arf-/- mice uniformly developed spontaneous OS by 7 months of age. Tax+Arf-/- tumors were well differentiated OS characterized by an abundance of new bone with OC recruitment, expressed OB markers and displayed intact levels of p53 mRNA and reduced Rb transcript levels. Cell lines established from OS recapitulated characteristics of the primary tumor, including the expression of mature OB markers and ability to form mineralized tumors when transplanted. Loss of heterozygosity in OS tumors arising in Tax+Arf+/- mice emphasized the necessity of ARF-loss in OS development. Hypothesizing that inhibition of ARF-regulated bone remodeling would repress development of OS, we demonstrated that treatment of Tax+Arf-/- mice with zoledronic acid, a bisphosphonate inhibitor of OC activity and repressor of bone turnover, prevented or delayed the onset of OS. These data describe a novel role for ARF as a regulator of bone remodeling through effects on both OB and OC. Finally, these data underscore the potential of targeting bone remodeling as adjuvant therapy or in patients with genetic predispositions to prevent the development of OS

Transforming growth factor-[beta]1 regulates steady-state PTH/PTHrP receptor mRNA levels and PTHrP binding in ROS 17/2.8 osteosarcoma cells

The effect of transforming growth factor [beta]1 (TGF-[beta]1) on the expression of mRNA for the parathyroid hormone receptor and binding of iodinated parathyroid hormone-related protein in ROS 17/2.8 osteosarcoma cells was evaluated. TGF-[beta]1 stimulated a 2-7-fold increase in steady state mRNA levels for the parathyroid hormone receptor at a maximal dose of 5 ng/ml, with increased levels of expression at 6 h of TGF-[beta]1-incubation, and peak levels at 8-24 h. Receptor binding studies revealed a significant increase in PTHrP-specific binding with TGF-[beta]1 doses as low as 0.5 ng/ml and a 55% increase in numbers of receptors with no alteration in binding affinity with 5.0 ng/ml TGF-[beta]1. Time course studies indicated that receptor binding was increased at 24 h with peak levels reached at 48 h of treatment. PTH-stimulated cAMP levels were significantly increased in ROS 17/2.8 cells treated with TGF-[beta]1 (0.5 ng/ml) for 48 h. These data indicate that TGF-[beta]1 upregulates steady-state mRNA, ligand binding and PTH/PTHrP receptor signaling in rat osteosarcoma cells. The effects of TGF-[beta]1 on bone may be attributed in part to regulation of the PTH/PTHrP receptor at the molecular level.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31617/1/0000549.pd

Secretion of parathyroid hormone-related protein by bovine mammary cells in vitro

Mammary cells were isolated from lactating cows at 1 to 6 weeks after calving and evaluated for their ability to secrete PTHrP in vitro. The tissue was enzymatically digested to release glandular acini. The digested acini were cultured on thin (1.0 mm) or thick (2.5 mm) layers of collagen. The cultures containing thick collagen were detached and allowed to contract on day 6. The culture medium consisted of M199 with prolation (8 µg/ml), insulin (5 µg/ml), cortisol (5 µg/ml), and fetal bovine serum (15%). PTHrP production was measured by N-terminal RIA and bioassay (stimulation of adenylate cyclase in the ROS 17/2.8 cell line). Medium was collected at 2-day intervals for 14 days. The cells reached confluence at 4–6 days. PTHrP production was low at day 2 (<0.5 ng/ml), but increased to peak production (2–4 ng/ml) at approximately day 6–8 of culture and remained constant until day 14. Immunoreactive and bioactive PTHrP levels in the culture medium correlated well. The cultures produced high levels of lactoferrin (500 to 3000 ng/ml) and low levels of α s1 -casein (14 to 77 ng/ml).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41592/1/774_2006_Article_BF02375695.pd

Cellular mechanisms of bone resorption in breast carcinoma

The cellular mechanisms that account for the increase in osteoclast numbers and bone resorption in skeletal breast cancer metastasis are unclear. Osteoclasts are marrow-derived cells which form by fusion of mononuclear phagocyte precursors that circulate in the monocyte fraction. In this study we have determined whether circulating osteoclast precursors are increased in number or have an increased sensitivity to humoral factors for osteoclastogenesis in breast cancer patients with skeletal metastases (± hypercalcaemia) compared to patients with primary breast cancer and age-matched normal controls. Monocytes were isolated and cocultured with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D3[1,25(OH)2D3] and human macrophage colony stimulating factor (M-CSF) on coverslips and dentine slices. Limiting dilution experiments showed that there was no increase in the number of circulating osteoclast precursors in breast cancer patients with skeletal metastases (± hypercalcaemia) compared to controls. Osteoclast precursors in these patients also did not exhibit increased sensitivity to 1,25(OH)2D3 or M-CSF in terms of osteoclast formation. The addition of parathyroid hormone-related protein and interleukin-6 did not increase osteoclast formation. The addition of the supernatant of cultured breast cancer cell lines (MCF-7 and MDA-MB-435), however, significantly increased monocyte-osteoclast formation in a dose-dependent fashion. These results indicate that the increase in osteoclast formation in breast cancer is not due to an increase in the number/nature of circulating osteoclast precursors. They also suggest that tumour cells promote osteoclast formation in the bone microenvironment by secreting soluble osteoclastogenic factor(s). © 2001 Cancer Research Campaign http://www.bjcancer.co