Mer Tyrosine Kinase Regulates Disseminated Prostate Cancer Cellular Dormancy.

Many prostate cancer (PCa) recurrences are thought to be due to reactivation of disseminated tumor cells (DTCs). We previously found a role of the TAM family of receptor tyrosine kinases TYRO3, AXL, and MERTK in PCa dormancy regulation. However, the mechanism and contributions of the individual TAM receptors is largely unknown. Knockdown of MERTK, but not AXL or TYRO3 by shRNA in PCa cells induced a decreased ratio of P-Erk1/2 to P-p38, increased expression of p27, NR2F1, SOX2, and NANOG, induced higher levels of histone H3K9me3 and H3K27me3, and induced a G1/G0 arrest, all of which are associated with dormancy. Similar effects were also observed with siRNA. Most importantly, knockdown of MERTK in PCa cells increased metastasis free survival in an intra-cardiac injection mouse xenograft model. MERTK knockdown also failed to inhibit PCa growth in vitro and subcutaneous growth in vivo, which suggests that MERTK has specificity for dormancy regulation or requires a signal from the PCa microenvironment. The effects of MERTK on the cell cycle and histone methylation were reversed by p38 inhibitor SB203580, which indicates the importance of MAP kinases for MERTK dormancy regulation. Overall, this study shows that MERTK stimulates PCa dormancy escape through a MAP kinase dependent mechanism, also involving p27, pluripotency transcription factors, and histone methylation. J. Cell. Biochem. 118: 891-902, 2017. © 2016 Wiley Periodicals, Inc.

A novel method for monitoring tumor proliferation in vivo using fluorescent dye DiD.

Monitoring single cell proliferation in vivo is difficult, but optimizing this technique is essential in order to expand our knowledge of the regulation of tumor proliferation. In this study, we used a lipophilic fluorescent dye, DiD, that rapidly and stably integrates into the phospholipid cell membrane. We cultured DiD-stained prostate cancer cell lines for 10 days and isolated cells by flow cytometry based on expression levels of DiD. We found that a decrease in DiD intensity was correlated to the reduction of EdU, where the DiD-high population proliferated more slowly than the DiD-low population and the DiD-low population exhibited a higher mitotic index. We also found that DiD was detected after 3 weeks of implantation in an in vivo setting. Importantly, DiD dye did not have any effect on normal cell growth, whereas a gold standard fluorescent dye for measuring cell proliferation, CFSE, slowed cell proliferation. Although further study is indicated, DiD can be useful for identifying the molecular mechanisms underlying tumor proliferation in vivo.

Erythropoietin supports the survival of prostate cancer, but not growth and bone metastasis.

Erythropoietin (Epo) is used in clinical settings to enhance hematopoietic function and to improve the quality of life for patients undergoing chemotherapy by reducing fatigue and the need for transfusions. However, several meta-analyses have revealed that Epo treatments are associated with an increased risk of mortality in cancer patients. In this study, we examined the role of Epo in prostate cancer (PCa) progression, using in vitro cell culture systems and in vivo bone metastatic assays. We found that Epo did not stimulate the proliferation of PCa cell lines, but did protect PCa cells from apoptosis. In animal models of PCa metastasis, no evidence was found to support the hypothesis that Epo enhances metastasis. Together, these findings suggest that Epo may be useful for treating severe anemia in PCa patients without increasing metastatic risk.

Proteoglycan 4, a novel immunomodulatory factor, regulates parathyroid hormone actions on hematopoietic cells.

Proteoglycan 4 (PRG4), a critical protective factor in articular joints, is implicated in hematopoietic progenitor cell expansion and megakaryopoiesis. PRG4 loss-of-function mutations result in camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome, which is characterized primarily by precocious joint failure. PRG4 was identified as a novel parathyroid hormone (PTH) responsiveness gene in osteoblastic cells in bone, and was investigated as a potential mediator of PTH actions on hematopoiesis. Sixteen-week-old Prg4(-/-) mutant and Prg4(+/+) wild-type mice were treated daily with intermittent PTH (residues 1-34) or vehicle for 6 weeks. At 22 weeks of age, Prg4 mutant mice had increased peripheral blood neutrophils and decreased marrow B220(+) (B-lymphocytic) cells, which were normalized by PTH. The PTH-induced increase in marrow Lin(-)Sca-1(+)c-Kit(+) (hematopoietic progenitor) cells was blunted in mutant mice. Basal and PTH-stimulated stromal cell-derived factor-1 (SDF-1) was decreased in mutant mice, suggesting SDF-1 as a candidate regulator of proteoglycan 4 actions on hematopoiesis in vivo. PTH stimulation of IL-6 mRNA was greater in mutant than in wild-type calvaria and bone marrow, suggesting a compensatory mechanism in the PTH-induced increase in marrow hematopoietic progenitor cells. In summary, proteoglycan 4 is a novel PTH-responsive factor regulating immune cells and PTH actions on marrow hematopoietic progenitor cells.

Tumor expressed PTHrP facilitates prostate cancer-induced osteoblastic lesions.

Expression of parathyroid hormone-related protein (PTHrP) correlates with prostate cancer skeletal progression; however, the impact of prostate cancer-derived PTHrP on the microenvironment and osteoblastic lesions in skeletal metastasis has not been completely elucidated. In this study, PTHrP overexpressing prostate cancer clones were stably established by transfection of full length rat PTHrP cDNA. Expression and secretion of PTHrP were verified by western blotting and IRMA assay. PTHrP overexpressing prostate cancer cells had higher growth rates in vitro, and generated larger tumors when inoculated subcutaneously into athymic mice. The impact of tumor-derived PTHrP on bone was investigated using a vossicle co-implant model. Histology revealed increased bone mass adjacent to PTHrP overexpressing tumor foci, with increased osteoblastogenesis, osteoclastogenesis and angiogenesis. In vitro analysis demonstrated pro-osteoclastic and pro-osteoblastic effects of PTHrP. PTHrP enhanced proliferation of bone marrow stromal cells and early osteoblast differentiation. PTHrP exerted a pro-angiogenic effect indirectly, as it increased angiogenesis but only in the presence of bone marrow stromal cells. These data suggest PTHrP plays a role in tumorigenesis in prostate cancer, and that PTHrP is a key mediator for communication and interactions between prostate cancer and the bone microenvironment. Prostate cancer-derived PTHrP is actively involved in osteoblastic skeletal progression.

JunB as a downstream mediator of PTHrP actions in cementoblasts.

UNLABELLED: The role of AP-1 family members in the action of PTHrP was examined in cementoblasts. PTHrP increased mRNA and protein levels of all Fos members, but only one Jun member (JunB) was increased. Overexpression of JunB in cementoblasts mimicked actions of PTHrP to support osteoclastogenesis and inhibit cementoblast differentiation, suggesting that the actions of PTHrP on mesenchymal cells operate through JunB. INTRODUCTION: Cementoblasts are mesenchymal cells that share phenotypic features with osteoblasts in vitro; however, unlike osteoblasts, cementoblasts rarely support osteoclastogenesis in vivo. The osteoblast-mediated support of osteoclastogenesis involves PTH-induced reduction in osteoprotegerin (OPG) expression. PTH acts on osteoblastic cells through specific signaling pathways and transcription factors such as activator protein 1 (AP-1). The purpose of this study was to determine the impact of PTH-related protein (PTHrP) on AP-1 transcription factors in cementoblasts and the role of JunB in the actions of PTHrP. MATERIALS AND METHODS: Cementoblastic cells were treated with PTHrP and evaluated for mRNA and protein levels of AP-1 family members. Stable transfectants of OCCM cells overexpressing JunB were evaluated for OPG production, ability to support osteoclastogenesis, and measures of proliferation and differentiation. RESULTS: PTHrP treatment in vitro resulted in a time-dependent upregulation of mRNA and proteins for the Fos family members, but only JunB of the Jun family. OPG mRNA and protein levels were reduced by PTHrP in OCCM and were lower in JunB overexpressing cells than controls. In co-culture experiments, TRACP+ cells were increased with RANKL treatment in JunB overexpressing cells compared with controls. Cementoblast differentiation was reduced with overexpression of JunB as measured by a decrease in mineralized nodule formation and gene expression for bone sialoprotein and osterix. Measures of proliferation including cell number and cyclin D1 levels were increased in JunB overexpressing clones. In vivo, cementoblast implants exhibited a cementoblastoid nature with copious mineral-like matrix, whereas JunB-overexpressing implants were densely cellular with little mineralized matrix. CONCLUSIONS: JunB was the only Jun family member increased by PTHrP, and its overexpression showed similar patterns of gene expression and OPG production as PTHrP treatment of controls. These data suggest that JunB may be a key mediator of PTHrP actions in cementoblasts.

The marrow niche controls the cancer stem cell phenotype of disseminated prostate cancer.

Dissemination of cancer stem cells (CSCs) serves as the basis of metastasis. Recently, we demonstrated that circulating prostate cancer targets the hematopoietic stem cell (HSCs) 'niche' in marrow during dissemination. Once in the niche, disseminated tumor cells (DTCs) may remain dormant for extended periods. As the major function of the HSC niche is to maintain stem cell functions, we hypothesized that the niche regulates CSC activities of DTCs. Here we show that DTCs recovered from marrow were significantly enriched for a CSC phenotype. Critically, the conversion of DTCs to CSCs is regulated by niche-derived GAS6 through the Mer/mTOR; molecules previously shown to regulate dormancy. The data demonstrate that the niche plays a significant role in maintaining tumor-initiating prostate cancer in marrow and suggests a functional relationship between CSCs and dormancy. Understanding how the marrow niche regulates the conversion of DTCs to CSCs is critical for the development of therapeutics specifically targeting skeletal bone metastasis and dormancy.

PTHrP signaling targets cyclin D1 and induces osteoblastic cell growth arrest.

UNLABELLED: PTHrP control of the MC3T3-E1 cell cycle machinery showed that, during differentiation, PTHrP induced G1 growth arrest. Cyclin D1 was a critical mediator as a downstream effector of cAMP, PKC, and MAPK signaling, and the process was PKA-independent. The involvement of JunB has been found critical for PTHrP effects. INTRODUCTION: PTH-related protein (PTHrP) has been implicated in the control of bone cell turnover, but the mechanisms underlying its effect on osteoblast proliferation and differentiation have not been clearly defined. The mechanisms by which PTHrP impacts cell cycle proteins and the role of signaling pathways in differentiated osteoblasts were studied. MATERIALS AND METHODS: To elucidate the role of PTHrP, flow cytometric analyses were performed using MC3T3-E1 and primary mouse calvarial cells. Relative protein abundance (Western blot), physical association of partners (immunoprecipitation), and kinase activities (in vitro kinase assays using either GST-Rb or H1-histone as substrates) of cell cycle-associated proteins in vehicle and PTHrP-treated 7-day differentiated cells were determined. ELISA and/or Northern blot analyses were done to evaluate JunB and cyclin D1 expression. SiRNA-mediated gene silencing experiments were performed to silence JunB protein. Finally, inhibitors of cAMP, protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) were used to determine involvement of different signaling pathways. RESULTS: PTHrP inhibited cyclin D1 protein expression 7-fold in a dose- and time-dependent manner and increased the level of p16 protein in differentiated osteoblasts. Additionally, PTHrP reduced cyclin D1-CDK4/CDK6 and CDK1 kinase activities. Forskolin, a cAMP agonist, mimicked PTHrP action, and the PKC inhibitor, GF109203X, slightly blocked downregulation of cyclin D1, implying involvement of both cAMP and PKC. U0126, a MAPK inhibitor, alone decreased cyclin D1 protein, suggesting that the basal cyclin D1 protein is MAPK dependent. H-89, a PKA inhibitor, did not alter the effect of PTHrP on cyclin D1, suggesting a PKA-independent mechanism. Finally, expression of JunB, an activating protein-1 transcription factor, was significantly upregulated, and silencing JunB (siRNA) partially reversed the cyclin D1 response, implying involvement of JunB in the PTHrP-mediated growth arrest of MC3T3-E1 cells. CONCLUSION: PTHrP upregulates JunB and reduces cyclin D1 expression while inducing G1 cell cycle arrest in differentiated osteoblasts. Such regulation could be an important determinant of the life span and bone-forming activity of osteoblasts.

Bisphosphonate modulates cementoblast behavior in vitro.

BACKGROUND: Cementum formation is deemed to be instrumental for the successful regeneration of periodontal tissues, and thus events and modifiers of cementum formation and mineralization need to be determined. This study aimed to determine whether the bisphosphonate 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) altered the behavior of immortalized cementoblasts (osteocalcin-cementoblasts [OCCM]). METHODS: OCCM from transgenic mice were exposed to HEBP at concentrations ranging from 0.01 to 10.0 microM. The assays performed included the count of cell number for proliferation, Northern blot analysis for gene expression (up to 10 days for core binding factor alpha-1 [Cbfa1], bone sialoprotein [BSP], osteocalcin [OCN], and osteopontin [OPN], markers for cementoblast/osteoblast maturation/mineralization), von Kossa stain and alizarin red S stain for mineralization, and enzyme assay (p-nitrophenol phosphate cleavage) for alkaline phosphatase (ALP) activity. RESULTS: Mineral nodule formation was inhibited at the higher doses of HEBP (1.0 and 10.0 microM) only. At early stages (1, 3, and 6 days), gene expression assays revealed only subtle changes in treated cells versus untreated cells, but by day 10, groups treated with lower doses (0.01 and 0.1 microM) were markedly different at the gene expression level. OCN was significantly downregulated (70%) at the lowest dose, with less pronounced effects at higher doses. In concurrence, the master switch gene for osteoblasts, Cbfa1, was also downregulated at the lower doses. Inversely, OPN mRNA was enhanced at the lower doses. ALP activity was not altered by HEBP. CONCLUSION: Bisphosphonate alters cementoblast function in vitro through the regulation of gene expression and mineral formation.

Dexamethasone and basic-fibroblast growth factor regulate markers of mineralization in cementoblasts in vitro.

BACKGROUND: The aim of this study was to determine the effects of basic-fibroblast growth factor (b-FGF) and/or dexamethasone (Dex) on cementoblasts in vitro. METHODS: Murine cementoblasts were treated as follows: 1) 5% FBS (fetal bovine serum) + ascorbic acid (AA, 50 microg/ml, control); 2) 5% FBS + Dex (10(7)M) + AA; 3) 5% FBS + b-FGF (50 ng/ml)+AA; or 4) 5% FBS + Dex (10(7) M) + b-FGF (50 ng/ml)+AA and then evaluated by Northern analysis for changes in specific genes and by von Kossa stain for changes in mineral nodule formation. RESULTS: Mitotic activity: b-FGF stimulated DNA synthesis significantly versus negative control. Gene expression: osteocalcin (OCN): Dex or b-FGF or the combination resulted in a decrease in expression versus control. Bone sialoprotein (BSP): Dex increased expression of BSP mRNA levels, b-FGF decreased transcript for BSP at 6 and 24 hours. Long-term (8 days) Dex, b-FGF, or Dex plus b-FGF caused a decrease in BSP expression versus control; osteopontin (OPN): both Dex and b-FGF increased transcripts for OPN seen by 6 hours, with a greater increase noted with b-FGF versus Dex. No apparent additive effect of Dex with b-FGF was noted; matrix gamma-carboxyglutamic acid protein (MGP): b-FGF induced transcripts for MGP and addition of Dex increased this effect, while Dex alone had no effect on expression. Biomineralization: Dex increased cementoblast- mediated biomineralization, while b-FGF blocked this activity, and addition of Dex to b-FGF did not alter FGF associated inhibition. CONCLUSION: Dex and FGF alone and in combination alter cementoblast behavior, but additional studies are required to determine whether these factors have beneficial effects at the clinical level.

Bone marrow as a metastatic niche for disseminated tumor cells from solid tumors.

Bone marrow is a heterogeneous organ containing diverse cell types, and it is a preferred metastatic site for several solid tumors such as breast and prostate cancer. Recently, it has been shown that bone metastatic cancer cells interact with the bone marrow microenvironment to survive and grow, and thus this microenvironment is referred to as the 'metastatic niche'. Once cancer cells spread to distant organs such as bone, the prognosis for the patient is generally poor. There is an urgent need to establish a greater understanding of the mechanisms whereby the bone marrow niche influences bone metastasis. Here we discuss insights into the contribution of the bone marrow 'metastatic niche' to progression of bone metastatic disease, with a particular focus on cells of hematopoietic and mesenchymal origin.

Tissue engineering a surrogate niche for metastatic cancer cells.

In breast and prostate cancer patients, the bone marrow is a preferred site of metastasis. We hypothesized that we could use tissue-engineering strategies to lure metastasizing cancer cells to tissue-engineered bone marrow. First, we generated highly porous 3D silk scaffolds that were biocompatible and amenable to bone morphogenetic protein 2 functionalization. Control and functionalized silk scaffolds were subcutaneously implanted in mice and bone marrow development was followed. Only functionalized scaffolds developed cancellous bone and red bone marrow, which appeared as early as two weeks post-implantation and further developed over the 16-week study period. This tissue-engineered bone marrow microenvironment could be readily manipulated in situ to understand the biology of bone metastasis. To test the ability of functionalized scaffolds to serve as a surrogate niche for metastasis, human breast cancer cells were injected into the mammary fat pads of mice. The treatment of animals with scaffolds had no significant effect on primary tumor growth. However, extensive metastasis was observed in functionalized scaffolds, and the highest levels for scaffolds that were in situ manipulated with receptor activator of nuclear factor kappa-B ligand (RANKL). We also applied this tissue-engineered bone marrow model in a prostate cancer and experimental metastasis setting. In summary, we were able to use tissue-engineered bone marrow to serve as a target or "trap" for metastasizing cancer cells.

Annexin 2-CXCL12 interactions regulate metastatic cell targeting and growth in the bone marrow.

UNLABELLED: Annexin 2 (ANXA2) plays a critical role in hematopoietic stem cell (HSC) localization to the marrow niche. In part, ANXA2 supports HSCs by serving as an anchor for stromal-derived factor-1 (CXCL12/SDF-1). Recently, it was demonstrated that prostate cancer cells, like HSCs, use ANXA2 to establish metastases in marrow. The present study determined the capacity of ANXA2 expression by bone marrow stromal cells (BMSC) to facilitate tumor recruitment and growth through ANXA2-CXCL12 interactions. Significantly more CXCL12 was expressed by BMSC(Anxa2) (+/+) than by BMSC(Anxa2) (-/-) resulting in more prostate cancer cells migrating and binding to BMSC(Anxa2) (+/+) than BMSC(Anxa2) (-/-), and these activities were reduced when CXCL12 interactions were blocked. To further confirm that BMSC signaling through ANXA2-CXCL12 plays a critical role in tumor growth, immunocompromised SCID mice were subcutaneously implanted with human prostate cancer cells mixed with BMSC(Anxa2) (+/+) or BMSC(Anxa2) (-/-). Significantly larger tumors grew in the mice when the tumors were established with BMSC(Anxa2) (+/+) compared with the tumors established with BMSC(Anxa2) (-/-). In addition, fewer prostate cancer cells underwent apoptosis when cocultured with BMSC(Anxa2) (+/+) compared with BMSC(Anxa2) (-/-), and similar results were obtained in tumors grown in vivo. Finally, significantly more vascular structures were observed in the tumors established with the BMSC(Anxa2) (+/+) compared with the tumors established with BMSC(Anxa2) (-/-). Thus, ANXA2-CXCL12 interactions play a crucial role in the recruitment, growth, and survival of prostate cancer cells in the marrow. IMPLICATIONS: The tumor microenvironment interaction between ANXA2-CXCL12 is critical for metastatic phenotypes and may impact chemotherapeutic potential.

Bone morphogenetic protein 2 induces dental follicle cells to differentiate toward a cementoblast/osteoblast phenotype.

When triggered appropriately, dental follicle cells are considered to be able to differentiate toward a cementoblast/osteoblast phenotype. However, factors and mechanisms regulating follicle cell differentiation remain undefined. This study focused on determining the ability of bone morphogenetic protein (BMP) 2 to promote the differentiation of follicle cells and periodontal ligament (PDL) cells along a cementoblast/ osteoblast pathway. Follicle cells and PDL cells were isolated from the first molar region of CD-1 mice and immortalized with SV40. Both cell types expressed BMP-4 and BMP receptors (BMPR) IA and II, but only follicle cells expressed BMP-2 mRNA. Cells were exposed to recombinant human BMP (rhBMP)-2 (0-100 ng/ml) and Northern blots were used to determine the expression of mineral-associated markers. BMP-2, in a dose- and time-dependent manner, induced cementoblast/osteoblast differentiation of follicle cells, as reflected by enhanced core binding factor alpha (Cbfal), bone sialoprotein (BSP), and osteocalcin (OCN) mRNA expression and enhanced mineral formation. U0126, a specific inhibitor of MEK-1/2 members of the MAPK family, abolished BMP-2-mediated expression of BSP and OCN. In contrast, exposure of PDL cells to BMP-2 resulted in modest expression of OCN and minimal promotion of mineralization. These results suggest that BMP-2 triggers follicle cells to differentiate toward a cementoblast/osteoblast phenotype and that the MAPK pathway is involved.

The role of parathyroid hormone-related protein in the regulation of osteoclastogenesis by cementoblasts.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) promotes osteoclastogenesis by inhibiting expression of osteoprotegerin (OPG), a decoy receptor for the receptor activator of nuclear factor kappa B (RANK), and by enhancing production of RANK ligand (RANKL) by osteoblasts. However, little is known regarding the role of PTHrP in regulating cementoblast-mediated osteoclastogenesis. METHODS: This study determined the impact of PTHrP on osteoclastogenesis using: 1) OCCM-30 (immortalized murine cementoblasts), 2) RAW 264.7 cells (murine myeloid cells), or 3) OCCM-30 plus RAW 264.7 cells. Cells were treated with PTHrP (1-34), RANKL, or PTHrP and RANKL combined. Enzyme-linked immunosorbent assays (ELISAs) for OPG and RANKL were performed on media and cell lysates, and tartrate-resistant acid phosphatase (TRAP) and mRNA detection for the osteoclast associated receptor (OSCAR) were performed. RESULTS: The highest numbers of TRAP-positive cells and cells expressing OSCAR were found in the RAW cell group treated with either RANKL alone or RANKL and PTHrP. TRAP-positive cells were fewer when OCCM cells were co-cultured with RAW, but the greatest numbers were still with both PTHrP and RANKL. OPG levels were highest from OCCM cells and PTHrP decreased these levels. In contrast, RANKL levels were low in OCCM cell lysates and PTHrP increased RANKL. In vivo studies also revealed high osteoclastic activity surrounding developing teeth in mice administered PTH. CONCLUSIONS: These results demonstrate that PTHrP influences the balance of OPG and RANKL production by cementoblasts, and further indicate that this effect, in the context of surrounding cells, might have a significant impact on osteoclastogenesis, root resorption, and tooth eruption.

Cementoblast delivery for periodontal tissue engineering.

BACKGROUND: Predictable periodontal regeneration following periodontal disease is a major goal of therapy. The objective of this proof of concept investigation was to evaluate the ability of cementoblasts and dental follicle cells to promote periodontal regeneration in a rodent periodontal fenestration model. METHODS: The buccal aspect of the distal root of the first mandibular molar was denuded of its periodontal ligament (PDL), cementum, and superficial dentin through a bony window created bilaterally in 12 athymic rats. Treated defects were divided into three groups: 1) carrier alone (PLGA polymer sponges), 2) carrier + follicle cells, and 3) carrier + cementoblasts. Cultured murine primary follicle cells and immortalized cementoblasts were delivered to the defects via biodegradable PLGA polymer sponges, and mandibulae were retrieved 3 weeks and 6 weeks post-surgery for histological evaluation. In situ hybridization, for gene expression of bone sialoprotein (BSP) and osteocalcin (OCN), and histomorphometric analysis were further done on 3-week specimens. RESULTS: Three weeks after surgery, histology of defects treated with carrier alone indicated PLGA particles, fibrous tissue, and newly formed bone scattered within the defect area. Defects treated with carrier + follicle cells had a similar appearance, but with less formation of bone. In contrast, in defects treated with carrier + cementoblasts, mineralized tissues were noted at the healing site with extension toward the root surface, PDL region, and laterally beyond the buccal plate envelope of bone. No PDL-bone fibrous attachment was observed in any of the groups at this point. In situ hybridization showed that the mineralized tissue formed by cementoblasts gave strong signals for both BSP and OCN genes, confirming its nature as cementum or bone. The changes noted at 3 weeks were also observed at 6 weeks. Cementoblast-treated and carrier alone-treated defects exhibited complete bone bridging and PDL formation, whereas follicle cell-treated defects showed minimal evidence of osteogenesis. No new cementum was formed along the root surface in the above two groups. Cementoblast-treated defects were filled with trabeculated mineralized tissue similar to, but more mature, than that seen at 3 weeks. Furthermore, the PDL region was maintained with well-organized collagen fibers connecting the adjacent bone to a thin layer of cementum-like tissue observed on the root surface. Neoplastic changes were observed at the superficial portions of the implants in two of the 6-week cementoblast-treated specimens, possibly due in part to the SV40-transformed nature of the implanted cell line. CONCLUSIONS: This pilot study demonstrates that cementoblasts have a marked ability to induce mineralization in periodontal wounds when delivered via polymer sponges, while implanted dental follicle cells seem to inhibit periodontal healing. These results confirm the selective behaviors of different cell types in vivo and support the role of cementoblasts as a tool to better understand periodontal regeneration and cementogen-

Leucine-rich amelogenin peptide: a candidate signaling molecule during cementogenesis.

BACKGROUND: Cementum is a critical mineralized tissue; however, control of its formation remains undefined. One hypothesis is that enamel matrix proteins/peptides secreted by ameloblasts and/or epithelial rest cells contribute to the control of cementum formation via epithelial-mesenchymal interactions. Here, we focused on determining whether or not leucine-rich amelogenin peptide (LRAP), translated from an alternatively spliced amelogenin RNA, altered cementoblast behavior. METHODS: Immortalized murine cementoblasts (OCCM-30) were exposed to LRAP and evaluated for: 1) proliferative activity; 2) gene expression using Northern blot for Cbfal (core binding factor alpha-1); OCN (osteocalcin), OPN (osteopontin), and real-time reverse transcription-polymerase chain reaction (RT-PCR) for OPG (osteoprotegerin); and RANKL (receptor activator of NF-kappaB ligand); 3) signaling pathway using inhibitors of PKA (THFA), PKC (GF109203X), and MAPK (UO126); and 4) mineralization evaluated by von Kossa and Alizarin-red. RESULTS: LRAP had no effect on cell proliferation up to 6 days, with a decrease in cell growth observed at the highest dose by 9 days versus untreated cells. LRAP down regulated OCN and up regulated OPN in a dose- and time-response fashion, and inhibited the capacity of mineral nodule formation. Transcripts for OPG were increased in LRAP-treated cells compared to control, but RANKL mRNA levels were not affected. Core binding factor alpha (Cbfa) mRNA, expressed constitutively, was not affected by LRAP. Signaling pathway assays suggested involvement of the MAPK pathway, since the addition of the MAPK inhibitor suppressed OPN expression in LRAP-treated cells. CONCLUSION: Leucine-rich amelogenin peptide appears to have a direct effect on cementoblast activity that may prove significant during development as well as in regeneration of periodontal tissues.

Amelogenin: a potential regulator of cementum-associated genes.

BACKGROUND: Studies suggest that enamel matrix proteins induce differentiation and mineralization of a variety of mesenchymal cells, including odontoblasts, osteoblasts, and cementoblasts. It has been postulated that this activity could be due to amelogenin-like proteins, known to be present in some mixtures of enamel matrix derivatives. Amelogenins have been reported to induce expression of a mineralized tissue-specific marker, bone sialoprotein (BSP), indicating that epithelial products can regulate the activity of mesenchyme-derived cells. METHODS: To explore the molecular mechanisms involved in BSP regulation, a clonal population of immortalized murine cementoblasts (OCCM-30) was exposed to full-length murine amelogenin protein (rp(H)M180), 0.1 microg/ml to 10.0 microg/ml, for 8 days in vitro. To further investigate the potential epithelial-mesenchymal interaction, an amelogenin knockout mouse model was used to examine expression of BSP and other markers, including Type I collagen, in tissue samples. RESULTS: The lowest dose of amelogenin slightly enhanced BSP expression, whereas at the highest dose, a dramatic decrease (three-fold) in BSP expression was observed. Parallel experiments showed a corresponding decrease in mineral nodule formation in vitro for cells treated with the higher dose of rp(H)M180. In situ hybridization and immunohistochemical analysis of sections from amelogenin null mice revealed a dramatic reduction in expression of BSP mRNA and protein in cementoblasts and surrounding osteoblasts in comparison to age-matched controls. In contrast, the expression of Type I collagen was not significantly different from controls. CONCLUSION: These data suggest that amelogenin may be a critical signaling molecule required for appropriate development of the periodontium.

Cementoblasts maintain expression of osteocalcin in the presence of mineral trioxide aggregate.

This study investigated the effects of mineral trioxide aggregate on cementoblast growth and osteocalcin production in tissue culture. For cellular morphology studies, cementoblasts on mineral trioxide aggregate, IRM, and amalgam were incubated for 48 h then fixed for scanning electron microscopic analysis. For gene expression on mineral trioxide aggregate and IRM, reverse transcriptase polymerase chain reaction was performed using primer sets for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, alkaline phosphatase, osteocalcin, and bone sialoprotein after 3 and 5 days. In vitro matrix protein expression was evaluated by confocal microscopy for the presence of osteocalcin on MTA after 7 and 12 days. Images were compared with controls to assess qualitative differences. Results suggest that mineral trioxide aggregate permits cementoblast attachment and growth and the production of mineralized matrix gene and protein expression. Our data indicates that mineral trioxide aggregate can be considered cementoconductive.

Molecular pathways: niches in metastatic dormancy.

Despite the best available treatments for primary tumors, cancer can return, even after a long disease-free interval. During this period, cancer cells are believed to lie dormant in either primary sites, metastatic sites, or independent sites like bone marrow, effectively escaping adjuvant cytotoxic treatments. To date, little is known about how these cells transition to dormancy, or how they are reactivated if cancer recurs. Recent studies have revealed the effects of tumor microenvironment or niche on the regulation of tumor dormancy via the signaling pathways of growth arrest-specific 6, bone morphogenetic protein 7, and TGFß1, and that the balance between activation of p38 MAPK and ERK MAPK plays a pivotal role in tumor dormancy. In this review, we discuss tumor dormancy from the perspective of the niche and consider potential therapeutic targets. Greater understanding of the mechanisms involved will help guide innovation in the care of patients with advanced cancer.