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Low complication rates have been reported when cancellous bone is taken from the proximal tibia. The current study was done to determine the volume of cancellous bone that can be harvested from the proximal tibia and to determine the risk for postoperative tibia plateau fracture. The average compressed volume that could be harvested from nine tibiae was 5.4 cc compared with a reported volume of 6.0 cc from the iliac crest. In eight cadavers, the proximal tibia was decancellated in eight tibias. The decancellated and the matching eight contralateral intact tibias were loaded to determine the force required to cause a tibial plateau fracture. The null hypothesis, that there is no difference between the decancellated and the intact tibias, could not be rejected. The current experimental study supports the clinical findings that a sufficient amount of cancellous bone can be harvested from the proximal tibial metaphysis and that the risk of postoperative fracture is not increased. Therefore, the proximal tibia is a reliable cancellous bone graft donor site for clinical practice.

Pancreatic cancer is often resistant to conventional chemotherapy. In this study, we examined the role of adenovirus-mediated overexpression of E2F-1 in inducing apoptosis and increasing the sensitivity of pancreatic cancer cells to chemotherapeutic agents. MIA PaCa-2 pancreatic head exocrine adenocarcinoma cells (mutant p53) were treated by mock infection or adenoviruses expressing beta-galactosidase or E2F-1 (Ad-E2F-1) alone or in combination with sublethal concentrations of each chemotherapeutic drug. Cell growth and viability were assessed at selected time points. Apoptosis was evaluated by flow cytometry, characteristic changes in cell morphology and poly (ADP-ribose) polymerase (PARP) cleavage. Western blot analysis was used to examine the expression of E2F-1 and Bcl-2 family member proteins and PARP cleavage. Western blot analysis revealed marked overexpression of E2F-1 at a multiplicity of infection (MOI) of 20 and 70. By 3 days after infection, Ad-E2F-1 treatment at an MOI of 70 resulted in approximately a 20-fold reduction in cell growth and 60% reduction in cell viability as compared to mock-infected cells. Cell cycle analysis, PARP cleavage and changes in cell morphology supported apoptosis as the mechanism of cell death in response to E2F-1. In order to test the efficacy of treatment with a combination of gene therapy and chemotherapy, we utilized concentrations of Ad-E2F-1 which reduced viability to 50% in combination with each chemotherapeutic agent. Cotreatment of the cells with E2F-1 virus and roscovitine (ROS) or etoposide resulted in an additive effect on cell growth inhibition and induction of apoptosis. Interestingly, 5-fluorouracil did not cooperate with Ad-E2F-1 in the mediation of tumor death or inhibition of cell growth. Immunoblotting for Bcl-2 family members revealed no significant changes in the expression levels of Bcl-2, Bcl X(L), Bax or Bak following gene or 'chemogene' therapy with E2F-1. However, a Bax cleavage product was noted which was substantially increased by cotreatment with ROS or etoposide. E2F-1 overexpression initiates apoptosis and suppresses growth in pancreatic MIA PaCa-2 cells in vitro. E2F-1-mediated apoptosis was not associated with significant changes in the expression of Bcl-2 family member proteins in these pancreatic cancer cells. ROS and etoposide, when combined with E2F-1 overexpression, induce apoptosis in an additive manner. This chemogene combination may provide a potentially useful therapeutic strategy for advanced pancreatic cancer.

BACKGROUND E2F-1 is a transcription factor that stimulates cellular proliferation and cell cycle progression from G1 to S-phase. Somewhat paradoxically, E2F-1 also has the properties of a tumor suppressor. Overexpression of E2F-1 has been shown to induce apoptosis in some cancer cells. In the current study, the effect of adenovirus-mediated E2F-1 gene transfer on human melanoma cell growth was investigated. METHODS Two human melanoma cell lines, SK-MEL-28 (wild-type p53) and SK-MEL-2 (mutant p53), were treated by mock infection, infection with a control vector expressing the β-galactosidase gene (Ad5CMV-LacZ), or infection with a vector expressing E2F-1 (Ad5CMV-E2F-1) at a multiplicity of infection of 100. Cell proliferation and viability were determined by WST-1 assay and trypan blue exclusion, respectively. Apoptosis was assessed by cell flow cytometry and confirmed by cell morphology, in situ terminal deoxynucleotidyl nick end labeling assay, and poly(ADP-ribose) polymerase cleavage assay. RESULTS Marked overexpression of E2F-1 was evident in both cell lines 24 hours after infection with Ad5CMVE2F-1 by Western blot analysis. E2F-1 overexpression resulted in growth inhibition and rapid loss of cell viability. Overexpression of E2F-1 also resulted in premature S-phase entry and G2 arrest at 24 hours followed by apoptotic cell death at 48 hours. After Ad5CMVE2F-1 infection, expression of Bax and Bak was unchanged, whereas Mcl-1 levels decreased markedly. In SK-MEL-28 cells, Bcl-XL levels also declined after E2F-1 expression. Bcl-2 was undetectable in SK-MEL-28 cells but was increased in SK-MEL-2 cells in response to E2F-1 overexpression. CONCLUSIONS Adenovirus-mediated E2F-1 gene transfer efficiently induces widespread apoptosis in human melanoma cells. E2F-1 overexpression induced apoptosis in cell lines containing wild-type and mutant p53, suggesting that this effect does not require wild-type p53 function. Anti-apoptotic proteins of the Bcl-2 family, notably Mcl-1 and Bcl-XL, may be involved in mediating the response to E2F-1. These data suggest that adenovirus-mediated E2F-1 gene therapy may be effective in the treatment of melanoma. Cancer 1999;86:2021–33. © 1999 American Cancer Society.

Polyethylene wear-induced osteolysis is the most significant primary factor limiting the life span of total joint arthroplasty. To reduce ultra-high-molecular-weight polyethylene (UHMWPE) particulate wear debris, highly cross-linked polyethylene (HXPE) bearings have been introduced in total hip arthroplasty (THA). In vitro hip simulator wear studies with HXPE have demonstrated a decrease in volumetric wear at the hip by 42% to 100% when compared with conventional metal-on-polyethylene bearings. Early to intermediate clinical results suggest that the in vivo wear properties of HXPE products are superior to those of conventional UHMWPE. Second-generation HXPE materials that utilize alternate cross-linking and free radical quenching techniques have been developed and propose to further minimize wear and oxidation.

E2F-1 and p53 are sequence specific transcription factors that are intimately involved in the regulation of the cell cycle. In addition to their role in cell cycle control, both E2F-1 and p53 have been identified as tumor suppressors and mediators of apoptosis. We have shown previously that adenoviral-mediated E2F-1 overexpression induces efficient apoptosis in colon adenocarcinoma cells. Previous reports have suggested that E2F-1 and p53 cooperate to mediate apoptosis and therefore, in this study, we examined the efficacy of combination gene therapy using adenovirus vectors expressing E2F-1 and p53 in human colon adenocarcinoma cell lines, HT-29 and SW620 (both mutant p53). Cells were treated by mock infection or infection with adenoviral vectors expressing b-galactosidase (LacZ), E2F-1, p53 or a combination of E2F-1 and p53. IC25 concentrations of each virus were estimated and used for each treatment in order to detect any synergistic or cooperative effects on tumor cell death in the combination therapy. By 5 days post infection, E2F-1-overexpressing cells exhibited growth inhibition and approximately 40-50% cell death in both cell lines. Co-expression of p53 with E2F-1 abrogated E2F-1-mediated growth inhibition and cell death. Cell cycle analysis revealed that overexpression of E2F-1 resulted in an accumulation of cells in G2/M phase, while overexpression of p53 resulted in a G1 phase accumulation. However, co-expression of E2F-1 and p53 counteracted each other as fewer cells accumulated in G1 and G2/M when compared to either p53 or E2F-1 alone. Furthermore, co-expression of p53 with E2F-1 resulted in decreased levels of E2F-1 protein expression. Mechanistically, upregulation of the CDK inhibitory protein, p21(WAF1/CIP1), was demonstrated in HT-29 cells following overexpression of either E2F-1, p53 or the combination E2F-1/p53 therapy. However, in SW620 cells, only the cells infected with Ad-p53 alone or in combination resulted in upregulation of p21(WAF1/CIP1). These results suggest that p53 and p21(WAF1/CIP1) may cooperate to inhibit the expression and activity of E2F-1. In conclusion, combination adenoviral vector-mediated E2F-1 and p53 gene transfer was not therapeutically advantageous in this in vitro model of human colon adenocarcinoma.

E2F -1 is a transcription factor that regulates cell cycle progression into S-phase. Deregulation of E2F-1 activity has been associated with cellular commitment to apoptosis. Also critical in the regulation of S-phase are the actions of the cyclin dependent kinases, Cdk2 and cdc2. Inhibition of these cyclin dependent kinases has been similarly associated with disrupting orderly S-phase progression and causing subsequent apoptosis in certain cancer cells. In this study, we examine the ability of adenovirus-mediated E2F-1 overexpression to induce apoptosis in human gastric carcinoma cells. Furthermore, we investigate the effect of the cyclin dependent kinase inhibitors, olomoucine and roscovitine, on E2F-1-mediated apoptosis in human gastric carcinoma cells. AGS and SNU-1 gastric adenocarcinoma cells were infected with adenoviral vectors expressing E2F-1 (Ad5CMVE2F-1) or control viruses expressing beta-galactosidase (Ad5CMVLacZ) or lacking a transgene (Ad5). Gastric adenocarcinoma cells were then independently treated with roscovitine or olomoucine. Finally, gastric adenocarcinoma cells were infected with the various adenoviral vectors in combination with roscovitine or olomoucine. E2F-1 overexpression resulted in an 85% reduction in cell viability at 72 h compared to controls. Combining E2F-1 overexpression with roscovitine resulted in >99% reduction in cell viability by 72 h. Overexpression of E2F-1 resulted in premature S-phase entry and G2/M arrest at 24 h, followed by apoptosis by 72 h. Combining E2F-1 overexpression with roscovitine resulted in an earlier G2/M arrest, followed by a more complete, widespread apoptotic response by 24 h. Caspase 3/CPP32 activation and PARP cleavage in response to E2F-1 overexpression, alone and in combination with roscovitine, implicate the caspase cascade in E2F-1-mediated apoptosis of gastric cancer cells. Bax levels also increased in response to E2F-1 gene transfer, alone and in combination with roscovitine. E2F-1 overexpression induces widespread apoptosis in human gastric carcinoma cells. Combining E2F-1 overexpression with cyclin-dependent kinase inhibitors results in an enhanced apoptotic response, causing nearly complete gastric tumor cell death within 72 h. E2F-1 gene therapy in combination with cyclin dependent kinase inhibitors is a potentially active chemogene therapy strategy for the treatment of human gastric cancer.