Viable cryopreserved human bone graft exhibit superior osteogenic properties in mandibular lateral augmentation.

Lack of bone volume to place dental implants is frequently a problem in the reconstruction of edentulous patients. Even though autografts are the gold standard for jaw regeneration, morbidity associated with the harvesting site stimulates the demand for other substitutes. The aim of this study is to characterize the incorporation and the osteogenic ability of a viable cryopreserved human bone graft (VC-HBG) in the mandibular augmentation in rats. Bone chips from fresh human vertebrae cadaveric donors were processed, cryoprotected and deep-frozen at - 80 °C maintaining its cell viability. A jaw augmentation model was used in 20 athymic nude rats allocated into 2 groups to either receive the VC-HBG or an acellular graft as control (A-HBG). The assessment of the grafts' incorporation was performed at 4 and 8 weeks by micro-CT, histomorphometry and immunohistochemistry. Bone volume gain was significantly higher for the VC-HBG group at both time points. At 4 weeks, the A-HBG group presented significantly higher mineral density, but at 8 weeks, the VC-HBG group showed significantly higher values than the A-HBG. There was no statistical difference between VC-HBG and A-HBG groups at 4-weeks for remaining graft particles, while at 8 weeks, the VC-HBG group showed significantly less graft remnants. Collagen I, osteopontin and tartrate-resistant acid phosphatase expression were significantly higher in the VC-HBG group at both time points, while osteocalcin expression was significantly higher in the VC-HBG group at 8-weeks compared to the A-HBG group. This experimental research demonstrated that the VC-HBG shows positive osteogenic properties, greater bone formation, higher rate of bone remodeling and a better overall incorporation in rats' mandibles compared to the A-HBG.

Human Bone Marrow-Derived Mesenchymal Stromal Cell-Seeded Bone Biomaterial Directs Fast and Superior Mandibular Bone Augmentation in Rats.

Atrophic maxillary ridges present a challenge in the field of oral implantology. Autologous bone is still considered the gold standard grafting material, but the increased morbidity and surgical complications represent a major drawback for its use. The aim of this study was to assess the efficacy of an off-the-shelf cell-seeded bone biomaterial for mandibular bone augmentation, compared to its acellular counterpart. We used a rat model to test the osteogenic properties of bone marrow-derived mesenchymal stromal cells (MSCs)-seeded bone microparticles compared to acellular bone microparticles alone. Rats were euthanized at 4 and 8 weeks, and results analyzed using micro-CT imaging, histology (H&E, Masson's Trichrome), histomorphometry and immunohistology (Tartrate-Resistant Acid Phosphatase-TRAP, Osteocalcin and human specific anti-mitochondria antibodies). Micro-CT analysis demonstrated that the cell-seeded biomaterial achieved significantly more bone volume formation at 4 weeks (22.75 ± 2.25 mm3 vs 12.34 ± 2.91 mm3, p = 0.016) and at 8 weeks (64.95 ± 5.41 mm3 vs 42.73 ± 10.58 mm3, p = 0.029), compared to the acellular bone microparticles. Histology confirmed that the cell-seeded biomaterial was almost completely substituted at 8 weeks, in opposition to the acellular biomaterial group. Immunohistochemical analysis showed a significantly higher number of TRAP and Osteocalcin positive cells at 4 weeks in the cell-seeded group compared to the acellular group, thereby demonstrating a higher rate of bone remodeling in the presence of MSCs. The grafted human cells remained viable and were detected up to at least 8 weeks, as observed using the human specific anti-mitochondria antibody. This off-the-shelf material available in unlimited quantities could therefore represent a significant advance in the field of mandibular bone augmentation by providing a larger volume of new bone formation in a shorter time.

Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells.

Prostate cancer (PCa), a leading cause of cancer-related death in men, becomes resistant to androgen deprivation therapy by inducing androgen receptor (AR) activity, which is known as castration-resistant PCa (CRPC). Enzalutamide is an approved drug that inhibits AR activity and increases overall survival. However, resistance to enzalutamide develops rapidly often by increasing AR activity, suggesting that new therapies are required for CRPC. We investigated whether betulinic acid (BA), a small molecule from plants that inhibits multiple deubiquitinases (DUBs), reduces AR, and selectively kills PCa cells, can provide an adjuvant strategy for CRPC. Our data indicated that BA reduced AR protein stability and mRNA expression, making it an attractive agent for CRPC. BA decreased AR mRNA possibly by inhibiting a histone 2A DUB thereby increasing ubiquitinated histone 2A, a transcriptional repressor. We identified multiple and specific DUBs inhibited by BA either in PCa cells or using recombinant DUBs. Similar results were obtained using another multi-DUB inhibitor WP1130, suggesting that these DUB inhibitors can decrease AR expression and increase PCa-specific death. Our results also suggest that combining multi-DUB inhibitors BA or WP1130 with enzalutamide may provide a novel strategy for CRPC by further decreasing AR expression and increasing apoptotic cell death.

Incorporation and Remodeling of Bone Block Allografts in the Maxillary Reconstruction: A Randomized Clinical Trial.

BACKGROUND: Severe alveolar atrophy often presents a challenge for the implant surgery. The significant lack of bone in the alveolar ridges may compromise the final restorations both from the aesthetic and functional standpoints. OBJECTIVES: To evaluate the behavior of bone block allografts for the maxillary augmentation and to investigate its incorporation, remodeling, and implant survival rates in two different healing time points. MATERIAL AND METHODS: Sixty-six consecutive patients (52 female/14 male, mean age: 57.95 ± 9.06 years old), presenting 113 atrophic alveolar ridges underwent maxillary augmentation with fresh-frozen allogeneic bone blocks from tibia. Patients were randomly assigned in two groups: Group 1-patients who would wait 4 months for implant placement after grafting, and Group 2-patients who would wait 6 months. Events of infection, suture dehiscence or mucosal perforation were recorded. Cone-beam computed tomography scans were compared volumetrically between the time of the grafting surgery and reentry procedure after incorporation. Biopsies were collected and subjected to histological, histomorphometric and immunehistochemical analysis. RESULTS: A total of 305 implants were placed in the reconstructed sites. The mean resorption rate in Group 1 (13.98% ± 5.59) was significantly lower than Group 2 (31.52% ± 6.31). The amount of calcified tissue, newly formed bone and remaining graft particles demonstrated no difference between groups. The samples showed evident immunolabeling for the podoplanin protein in both groups. The implants cumulative survival rate was 94.76%. CONCLUSIONS: The findings of the present study indicate that there is a significant difference regarding the resorption of the grafts when waiting 4 or 6 months before placing the implants, even though no difference was found in the histological, histomorphometric, and immunohistochemical features. Both 4-month and 6-months healing times are suitable for the implant placement.

Mcl-1 protects prostate cancer cells from cell death mediated by chemotherapy-induced DNA damage.

The anti-apoptotic protein Mcl-1 is highly expressed in castration-resistant prostate cancer (CRPC), resulting in resistance to apoptosis and association with poor prognosis. Although predominantly localized in the cytoplasm, there is evidence that Mcl-1 exhibits nuclear localization where it is thought to protect against DNA damage-induced cell death. The role of Mcl-1 in mediating resistance to chemotherapy-induced DNA damage in prostate cancer (PCa) is not known. We show in human PCa cell lines and in TRAMP, a transgenic mouse model of PCa, that the combination of the antimitotic agent ENMD-1198 (analog of 2-methoxyestradiol) with betulinic acid (BA, increases proteotoxic stress) targets Mcl-1 by increasing its proteasomal degradation, resulting in increased γH2AX (DNA damage) and apoptotic/necrotic cell death. Knockdown of Mcl-1 in CRPC cells leads to elevated γH2AX, DNA strand breaks, and cell death after treatment with 1198 + BA- or doxorubicin. Additional knockdowns in PC3 cells suggests that cytoplasmic Mcl-1 protects against DNA damage by blocking the mitochondrial release of apoptosis-inducing factor and thereby preventing its nuclear translocation and subsequent interaction with the cyclophilin A endonuclease. Overall, our results suggest that chemotherapeutic agents that target Mcl-1 will promote cell death in response to DNA damage, particularly in CRPC.

ABT-737, a small molecule Bcl-2/Bcl-xL antagonist, increases antimitotic-mediated apoptosis in human prostate cancer cells.

Castration-resistant prostate cancer (CRPC) expresses high levels of the anti-apoptotic proteins Bcl-2, Bcl-xL and Mcl-1, resulting in resistance to apoptosis and association with poor prognosis. Docetaxel, an antimitotic drug that is the first-line treatment strategy for CRPC, is known to provide a small survival benefit. However, docetaxel chemotherapy alone is not enough to counteract the high levels of Bcl-2/Bcl-xL/Mcl-1 present in CRPC. ABT-737 is a small molecule that binds to Bcl-2/Bcl-xL (but not Mcl-1) with high affinity and disrupts their interaction with pro-apoptotic Bax/Bak, thus enhancing apoptosis. Our results indicate that ABT-737 can sensitize androgen-dependent LNCaP and CRPC PC3 cells to docetaxel- and to the novel antimitotic ENMD-1198-mediated caspase-dependent apoptosis. CRPC DU145 cells, however, are more resistant to ABT-737 because they are Bax null and not because they express the highest levels of anti-apoptotic Mcl-1 (associated with ABT-737 resistance). Knockdown of Bax or Bak in LNCaP indicates that ABT-737-induced antimitotic enhancement of apoptosis is more dependent on the levels of Bax than Bak. Furthermore, we find that the ability of docetaxel to increase cyclin B1/Cdk1-mediated phosphorylation of Bcl-2/Bcl-xL and decrease Mcl-1 is required for ABT-737 to enhance apoptosis in PC3 cells, as determined by addition of Cdk1 inhibitor purvalanol A and expression of shRNA specific for cyclin B1. Overall, our data suggests that the high levels of anti-apoptotic proteins in Bax-expressing CRPC cells can be overcome by targeting Bcl-2/Bcl-xL with ABT-737 and Mcl-1 with antimitotics.

Histologic, biomechanical, and biological evaluation of fan-folded iliotibial band allografts for anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to thoroughly characterize the fan-folded iliotibial band (FITB) allograft and compare it with anterior tibialis tendons (ATs) and native anterior cruciate ligaments (ACLs) to determine whether it measures up to those tissues. METHODS: We compared the histologic structure, tensile strength to failure, creep, and stress-relaxation properties of FITBs with those of ATs and ACLs. In vitro cytotoxicity and biocompatibility of FITBs were also compared with ATs. RESULTS: No structural difference was observed between the tissues studied. FITB ultimate tensile strength (3,459 ± 939 N) was not significantly different (P > .9999) from ultimate tensile strength of ATs (3,357 ± 111 N) and was significantly greater (P = .0005) than that of ACLs (886 ± 254 N). No significant difference (P > .9999) was observed in the increase in length resulting from creep testing between FITBs (9.5 ± 3.0 mm) and ATs (9.7 ± 4.0 mm). During stress-relaxation testing, FITBs reached 181 ± 46 N, which was not significantly different (P > .9999) from ATs (166 ± 40 N). Finally, we showed that cytotoxicity of FITBs and ATs was negligible. In vitro biocompatibility of FITBs and ATs was very good, whereas FITBs had a higher propensity to favor the attachment and infiltration of cells that proliferated for at least 4 weeks on their contact. CONCLUSIONS: We found that FITBs, ACLs, and ATs shared a similar structure made of aligned collagen fibers. No significant difference was observed between FITB and AT ultimate tensile strength, creep, and stress-relaxation viscoelastic properties. Ultimate tensile strength to failure of ACLs was lower than that of FITBs and ATs, whereas ACLs were superior to both FITBs and ATs during creep and stress-relaxation testing. FITBs and ATs showed low cytotoxicity and excellent biocompatibility in vitro, with a somewhat higher propensity of FITBs to favor cell attachment and infiltration over time. CLINICAL RELEVANCE: This study suggests that FITBs have the potential to perform as well as ATs for ACL reconstruction.

Betulinic acid selectively increases protein degradation and enhances prostate cancer-specific apoptosis: possible role for inhibition of deubiquitinase activity.

Inhibition of the ubiquitin-proteasome system (UPS) of protein degradation is a valid anti-cancer strategy and has led to the approval of bortezomib for the treatment of multiple myeloma. However, the alternative approach of enhancing the degradation of oncoproteins that are frequently overexpressed in cancers is less developed. Betulinic acid (BA) is a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells, making it an attractive anti-cancer agent. Our results in prostate cancer suggested that BA inhibited multiple deubiquitinases (DUBs), which resulted in the accumulation of poly-ubiquitinated proteins, decreased levels of oncoproteins, and increased apoptotic cell death. In normal fibroblasts, however, BA did not inhibit DUB activity nor increased total poly-ubiquitinated proteins, which was associated with a lack of effect on cell death. In the TRAMP transgenic mouse model of prostate cancer, treatment with BA (10 mg/kg) inhibited primary tumors, increased apoptosis, decreased angiogenesis and proliferation, and lowered androgen receptor and cyclin D1 protein. BA treatment also inhibited DUB activity and increased ubiquitinated proteins in TRAMP prostate cancer but had no effect on apoptosis or ubiquitination in normal mouse tissues. Overall, our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in prostate cancer but not in normal cells and tissues may provide an effective non-toxic and clinically selective agent for chemotherapy.

Neuroprotective properties of marrow-isolated adult multilineage-inducible cells in rat hippocampus following global cerebral ischemia are enhanced when complexed to biomimetic microcarriers.

Cell-based therapies for global cerebral ischemia represent promising approaches for neuronal damage prevention and tissue repair promotion. We examined the potential of marrow-isolated adult multilineage-inducible (MIAMI) cells, a homogeneous subpopulation of immature human mesenchymal stromal cell, injected into the hippocampus to prevent neuronal damage induced by global ischemia using rat organotypic hippocampal slices exposed to oxygen-glucose deprivation and rats subjected to asphyxial cardiac arrest. We next examined the value of combining fibronectin-coated biomimetic microcarriers (FN-BMMs) with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) pre-treated MIAMI compared to EGF/bFGF pre-treated MIAMI cells alone, for their in vitro and in vivo neuroprotective capacity. Naïve and EGF/bFGF pre-treated MIAMI cells significantly protected the Cornu Ammonis layer 1 (CA1) against ischemic death in hippocampal slices and increased CA1 survival in rats. MIAMI cells therapeutic value was significantly increased when delivering the cells complexed with FN-BMMs, probably by increasing stem cell survival and paracrine secretion of pro-survival and/or anti-inflammatory molecules as concluded from survival, differentiation and gene expression analysis. Four days after oxygen and glucose deprivation and asphyxial cardiac arrest, few transplanted cells administered alone survived in the brain whereas stem cell survival improved when injected complexed with FN-BMMs. Interestingly, a large fraction of the transplanted cells administered alone or in complexes expressed ßIII-tubulin suggesting that partial neuronal transdifferentiation may be a contributing factor to the neuroprotective mechanism of MIAMI cells.

Human marrow-isolated adult multilineage-inducible (MIAMI) cells protect against peripheral vascular ischemia in a mouse model.

BACKGROUND AIMS: The treatment of peripheral vascular disease (PVD) with stem cells potentially offers a promising strategy. We tested marrow-isolated adult multilineage-inducible (MIAMI) cells to induce neovascularization in a mouse model of critical hindlimb ischemia (CLI). METHODS: CLI was induced in the right hindlimb of Balb/C mice. One million MIAMI cells, normally grown at 3% O2, were injected in the adductor muscle along the ischemic region. All animals (n = 11 per group) were immunosuppressed with cyclosporine daily for the entire period. Human foreskin fibroblast (HFF) cells and phosphate-buffered saline (PBS) were used as controls. Blood perfusion in the ischemic right and non-ischemic left hindlimbs was measured. RESULTS: Compared with animals receiving HFF cells or PBS, MIAMI cells significantly improved blood perfusion, necrosis and inflammation in the ischemic limb. A fraction of injected MIAMI cells expressed CD31 and von Willebrand factor (vWF). MIAMI cells in vitro, under pro-angiogenic growth conditions, differentiated into endothelial-like cells and expressed endothelial markers such as CD31 and vWF, determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and CD31 and kinase insert domain receptor (KDR), determined by immunofluorescence. Moreover, MIAMI cells formed vascular endothelial-like tubules in the presence of matrigel. Bioplex immunoassay analysis showed increased secretion of angiogenic/anti-inflammatory factors by the MIAMI cells under 3% O2 compared with 21% O2, including monocyte chemoattractant protein-1 (MCP-1), fractalkine (Ftk), growth-related oncogene (GRO), vascular endothelial growth factor (VEGF), interleukin (IL)-6 and IL-8. Furthermore, transcripts for anti-inflammatory molecules stanniocalcin-1 (STC-1) and tumor necrosis factor-α-stimulated gene 6 (TSG-6) were up-regulated several fold. CONCLUSIONS: MIAMI cells can be very useful for patients affected by CLI. MIAMI cells promote blood vessel formation and reduce inflammation and necrosis in ischemic tissue.

NF-kappaB activation enhances cell death by antimitotic drugs in human prostate cancer cells.

BACKGROUND: NF-kappaB is a transcription factor that promotes inhibition of apoptosis and resistance to chemotherapy. It is commonly believed that inhibition of NF-kappaB activity can increase sensitivity of cancer cells to chemotherapy. However, there is evidence that NF-kappaB activation can sensitize cells to apoptosis and that inhibition of NF-kappaB results in resistance to chemotherapy. In prostate cancer, it is not clear in the different cell types (androgen-dependent and castration-resistant) if activation or inhibition of NF-kappaB is required for stimulation of apoptosis by chemotherapy. RESULTS: Our data indicate that the response of prostate cancer (PC) cells to the antimitotic drugs docetaxel (Doc) and 2-methoxyestradiol (2ME2) is dependent on the levels of NF-kappaB activity. In androgen-dependent LNCaP cells, Doc and 2ME2 treatment increased the low basal NF-kappaB activity, as determined by Western blot analysis of phospho-IkappaBalpha/p65, NF-kappaB promoter reporter assays, and p65 localization. Treatment of LNCaP cells with parthenolide, a pharmacologic inhibitor of NF-kappaB, or introduction of dominant-negative IkappaBalpha, or an shRNA specific for p65, a component of the NF-kappaB heterodimer, blocked apoptosis induced by Doc and 2ME2. In castration-resistant DU145 and PC3 cells, Doc and 2ME2 had little effect on the high basal NF-kappaB activity and addition of parthenolide did not enhance cell death. However, the combination of Doc or 2ME2 with betulinic acid (BA), a triterpenoid that activates NF-kappaB, stimulated apoptosis in LNCaP and non-apoptotic cell death in DU145 and PC3 cells. Increased sensitivity to cell death mediated by the Doc or 2ME2 + BA combination is likely due to increased NF-kappaB activity. CONCLUSIONS: Our data suggest that the combination of antimitotic drugs with NF-kappaB inhibitors will have antagonistic effects in a common type of PC cell typical of LNCaP. However, combination strategies utilizing antimitotic drugs with BA, an activator of NF-kappaB, will universally enhance cell death in PC cells, notably in the aggressive, castration-resistant variety that does not respond to conventional therapies.

Changes in the expression of genes associated with intraneuronal amyloid-beta and tau in Alzheimer's disease.

The clinical hallmark of Alzheimer's disease (AD) is impairment of cognition associated with loss of synapses, accumulation of amyloid-beta (Abeta) both within neurons and as extracellular deposits, and neurofibrillary degeneration composed of phospho-tau. Neurons in the hippocampus are among those that are most vulnerable. The purpose of this study was to investigate the expression of genes associated with cognition, synapse, and mitochondrial function in hippocampal neurons of AD compared to normal individuals. Neurons from the hippocampus with intraneuronal Abeta immunoreactivity were captured with laser microdissection; RNA was extracted; and levels of brain-derived neurotrophic factor (BDNF), TrkB (BDNF receptor), dynamin-1 (DYN), and cytochrome C oxidase subunit II (COX2) were assessed with quantitative real-time polymerase chain reaction. We found no significant differences in the expression of these genes in AD between neurons associated with Abeta compared to those lacking Abeta immunoreactivity. Double immunofluorescence microscopy showed the number of hippocampal neurons coexpressing Abeta or phospho-tau and either BDNF, TrkB, or DYN was similar in AD and controls. Our results suggest that neither intraneuronal Abeta nor phospho-tau has obligatory effects on reducing the expression of genes important for memory and cognition in hippocampus of AD.

Molecular characterization of the Ggamma-globin-Tag transgenic mouse model of hormone refractory prostate cancer: comparison to human prostate cancer.

BACKGROUND: Prostate cancer (PrCa) has a high incidence in Western countries and at present, there is no cure for hormone refractory prostate cancer. Transgenic mouse models have proven useful for understanding mechanisms of prostate carcinogenesis. The characterization of genetically modified mouse PrCa models using high-throughput genomic analyses provides important information to guide appropriate experiment applications for such model. METHODS: We have analyzed the transcriptome of the hormone refractory and highly metastatic Fetal Globin-SV40/T-antigen (Ggamma-globin-Tag) transgenic mouse model for PrCa compared to normal mouse prostate tissue. Gene expression patterns found in Ggamma-globin-Tag mouse prostate tumors were compared with publicly available human localized and metastatic prostate tumors (GEO accession # GSE3325) through hierarchical cluster analysis, Pearson's rank correlation coefficient, and Self Organizing Feature Maps (SOM) analyses. RESULTS: Ggamma-globin-Tag tumors clustered closely with human metastatic tumors and gene expression patterns had a significant correlation (P < 0.01), unlike human localized primary tumors (P > 0.6). Bioinformatic analyses identified deregulated genetic pathways and networks in Ggamma-globin-Tag tumors, which displayed similarities to alterations in human PrCa. Changes in the expression of genes involved in DNA replication and repair (Rb1, p53, Myc, PCNA, DNMT3A) and growth factor signaling pathways (TGFbeta2, ERK1/2, NRas, and Notch1) are deregulated in the Ggamma-globin-Tag tumors, suggesting their key role in the oncogenic process. Identification of an enrichment of putative binding sites for transcription factors revealed eight transcription factors that may be important in Ggamma-globin-Tag carcinogenesis, including SP1, NF-Y, CREB, Elk1, and E2F. Novel genes related to microtubule regulation were also identified in Ggamma-globin-Tag tumors as potentially important candidate targets for PrCa. Overexpression of stathmin-1, whose expression was increased in human metastatic prostate tumors, was validated in Ggamma-globin-Tag tumors by immunohistochemistry. This protein belongs to the SV40/T-antigen cancer signature identified in previous studies in prostate, breast, and lung cancer mouse models. CONCLUSIONS: Our results show that the Ggamma-globin-Tag model for hormone refractory PrCa shares important features with aggressive, metastatic human PrCa. Given the role of stathmin-1 in the destabilization of microtubles and taxane resistance, the Ggamma-globin-Tag model and other SV40/T-antigen driven transgenic models may be useful for testing potential therapies directed at stathmin-1 in human prostate tumors.

Low dose combinations of 2-methoxyestradiol and docetaxel block prostate cancer cells in mitosis and increase apoptosis.

Clinical trials have shown that chemotherapy with docetaxel (Doc) combined with prednisone can improve survival of patients with androgen-independent prostate cancer (AI-PC). It is likely that the combination of Doc with other novel agents would also improve the survival of AI-PC patients. We investigated whether the combination of Doc and 2-methoxyestradiol (2ME2), an endogenous metabolite of estradiol promising for cancer therapy, can increase apoptotic cell death in prostate cancer cells. Low concentration 2ME2 (0.5-1 microM)+Doc (0.05-0.1 nM) combinations inhibit cell growth, increase G2/M cell cycle arrest, and increase apoptosis more effectively than the single concentrations in a variety of human AI-PC cells. Effects on apoptosis were associated with an increase in p53 protein and a decrease in cyclin A-dependent kinase activity. We then investigated whether the combination of 2ME2+Doc can increase apoptotic cell death and inhibit the growth of prostate tumors in the FG/Tag transgenic mouse model of AI-PC. Doses of 2ME2 and Doc that increase mitotic cell cycle arrest result in an increase in apoptosis and lower primary prostate tumor weights in FG/Tag mice. High dose 2ME2+Doc combinations did not increase G2/M cell cycle arrest or apoptosis in AI-PC cell lines and in the FG/Tag mice more than the single drugs. Overall, our data indicate that low dose 2ME2+Doc combinations may provide a treatment strategy that can improve therapeutic efficacy against AI-PC while reducing toxicity often seen in patients treated with Doc.

Progression of prostate cancer from a subset of p63-positive basal epithelial cells in FG/Tag transgenic mice.

Transgenic mice that allow targeting of SV40 T antigen (Tag) to the prostate provide a unique model to identify cancer-initiating cells and follow their progression from a normal cell phenotype into prostate cancer cells. We have developed the FG/Tag transgenic mouse model of prostate cancer using the human fetal globin (FG) promoter linked to Tag. Immunohistochemistry results show that before the development of prostate intraepithelial neoplasia (PIN), a subset of p63(+) basal epithelial cells expresses Tag. As in the case of human prostate cancer, there is a loss of p63(+) basal cells with neoplastic progression, and a long period of time is required for PIN lesions to develop into palpable prostate tumors. Other immunohistochemistry results show cellular heterogeneity in FG/Tag PIN lesions and primary tumors with neuroendocrine differentiation. Cell lines derived from primary prostate tumors showed characteristics of a neuroendocrine-epithelial intermediate cell type. The FG promoter has high transcriptional activity in intermediate (DU 145, PC-3) and p63(+) basal epithelial (LHSR-AR) prostate cancer cells. Therefore, the unexpected development of prostate cancer in the FG/Tag mice may be due to the presence of DNA elements in the FG promoter that can target Tag to specific basal or intermediate cells. We conclude that FG/Tag mouse is a unique model of prostate cancer because the initiating cells are a subset of p63(+) basal (possibly stem cells), which may be the true cells of origin for carcinogenesis in aggressive human prostate cancer.

Increased expression of cyclin B1 sensitizes prostate cancer cells to apoptosis induced by chemotherapy.

Chemotherapeutic drugs ideally should take advantage of the differences between transformed and normal cells and induce apoptosis only in cancer cells. One such difference may be the overexpression of cyclin B1 protein in cancer cells, which is required for the proper progression through mitosis. Previously, we showed that treatment of human prostate cancer cells with 2-methoxyestradiol (2-ME) or docetaxel results in an accumulation of cyclin B1 protein and an increase in cyclin B1 kinase activity, followed by induction of apoptotic cell death. Inhibition of cyclin B1 kinase lowers apoptosis induced by 2-ME and docetaxel. In this study, we established a positive correlation between cyclin B1 protein and apoptosis induced by chemotherapy in prostate cancer cells. There is minimal cyclin B1 and induction of apoptosis by chemotherapy in nontransformed cells. LNCaP and PC-3 prostate cancer cells stably overexpressing cyclin B1 are more sensitive to apoptosis induced by chemotherapy. LNCaP cells expressing cyclin B1 small interfering RNA to lower cyclin B1 protein or dominant negative cyclin-dependent kinase 1 to inhibit cyclin B1 kinase show a decrease in apoptosis. Increased sensitivity to apoptosis by overexpression of cyclin B1 may be due to lower Bcl-2, higher p53, and decreased neuroendocrine differentiation. We suggest that a cancer-specific mechanism whereby 2-ME and docetaxel may exert anti-prostate cancer activity is the deregulated activation of cyclin B1 kinase, leading to the induction of apoptotic cell death. Our results also suggest that higher levels of cyclin B1 in prostate cancer cells may be a good prognostic marker for chemotherapy.

Sequential combinations of flavopiridol and docetaxel inhibit prostate tumors, induce apoptosis, and decrease angiogenesis in the Ggamma/T-15 transgenic mouse model of prostate cancer.

BACKGROUND: We investigated whether sequential combinations of flavopiridol and docetaxel can increase apoptotic cell death and inhibit the growth of primary and metastatic prostate tumors in the Ggamma/T-15 transgenic mouse model of prostate cancer. METHODS: Transgenic males were treated and the weights of primary and metastatic prostate tumors determined. Immunohistochemistry and Western blot was performed to evaluate the differences in apoptosis, proliferation, and angiogenesis. RESULTS: Docetaxel was slightly more effective than flavopiridol in inhibiting primary prostate tumors, but neither drug alone inhibited metastases. Single drug treatments decreased angiogenesis but did not increase apoptosis. Both sequential combinations resulted in greater inhibition of primary and metastatic prostate tumors, increased apoptosis, and decreased angiogenesis compared to control mice. CONCLUSIONS: Flavopiridol and docetaxel sequence combinations were effective in inhibiting prostate tumors in the Ggamma/T-15 transgenic mice. An increase in apoptosis and a decrease in angiogenesis resulted in the greatest inhibition of prostate cancers.