Harm C. A. Graat

WEE1 inhibition sensitizes osteosarcoma to radiotherapy

BackgroundThe use of radiotherapy in osteosarcoma (OS) is controversial due to its radioresistance. OS patients currently treated with radiotherapy generally are inoperable, have painful skeletal metastases, refuse surgery or have undergone an intralesional resection of the primary tumor. After irradiation-induced DNA damage, OS cells sustain a prolonged G2 cell cycle checkpoint arrest allowing DNA repair and evasion of cell death. Inhibition of WEE1 kinase leads to abrogation of the G2 arrest and could sensitize OS cells to irradiation induced cell death.MethodsWEE1 expression in OS was investigated by gene-expression data analysis and immunohistochemistry of tumor samples. WEE1 expression in OS cell lines and human osteoblasts was investigated by Western blot. The effect of WEE1 inhibition on the radiosensitivity of OS cells was assessed by cell viability and caspase activation analyses after combination treatment. The presence of DNA damage was visualized using immunofluorescence microscopy. Cell cycle effects were investigated by flow cytometry and WEE1 kinase regulation was analyzed by Western blot.ResultsWEE1 expression is found in the majority of tested OS tissue samples. Small molecule drug PD0166285 inhibits WEE1 kinase activity. In the presence of WEE1-inhibitor, irradiated cells fail to repair their damaged DNA, and show higher levels of caspase activation. The inhibition of WEE1 effectively abrogates the irradiation-induced G2 arrest in OS cells, forcing the cells into premature, catastrophic mitosis, thus enhancing cell death after irradiation treatment.ConclusionWe show that PD0166285, a small molecule WEE1 kinase inhibitor, can abrogate the G2 checkpoint in OS cells, pushing them into mitotic catastrophe and thus sensitizing OS cells to irradiation-induced cell death. This suggests that WEE1 inhibition may be a promising strategy to enhance the radiotherapy effect in patients with OS.

Prosthetic joint-associated infections treated with DAIR (debridement, antibiotics, irrigation, and retention) Analysis of risk factors and local antibiotic carriers in 91 patients

Background and purpose For prosthetic joint-associated infection (PJI), a regimen of debridement, antibiotics, irrigation, and retention of the prosthesis (DAIR) is generally accepted for acute infections. Various risk factors associated with treatment success have been described. The use of local antibiotic carriers (beads and sponges) is relatively unknown. We retrospectively analyzed risk factors in a cohort of patients from 3 hospitals, treated with DAIR for PJI. Patients and methods 91 patients treated with DAIR for hip or knee PJI in 3 Dutch centers between 2004 and 2009 were retrospectively evaluated. The mean follow-up was 3 years. Treatment success was defined as absence of infection after 2 years, with retention of the prosthesis and without the use of suppressive antibiotics. Results 60 patients (66%) were free of infection at follow-up. Factors associated with treatment failure were: a history of rheumatoid arthritis, late infection (> 2 years after arthroplasty), ESR at presentation above 60 mm/h, and infection caused by coagulase-negative Staphylococcus. Symptom duration of less than 1 week was associated with treatment success. The use of gentamicin sponges was statistically significantly higher in the success group, and the use of beads was higher in the failure group in the univariate analysis, but these differences did not reach significance in the logistic regression analysis. Less surgical procedures were performed in the group treated with sponges than in the group treated with beads. Interpretation In the presence of rheumatoid arthritis, duration of symptoms of more than 1 week, ESR above 60 mm/h, late infection (> 2 years after arthroplasty), and coagulase-negative Staphylococcus PJI, the chances of successful DAIR treatment decrease, and other treatment methods should be considered.

Different susceptibility of osteosarcoma cell lines and primary cells to treatment with oncolytic adenovirus and doxorubicin or cisplatin

Despite improvements in treatment regimens for osteosarcoma (OS) patients, survival rate has not increased over the last two decades. New treatment modalities are therefore warranted. Preclinical results with conditionally replicative adenoviruses (CRAds) to treat OS are promising. One type of CRAd that was effective against OS cells is Ad5-Δ24RGD. In other types of cancer, CRAds have been shown to interact synergistically with chemotherapeutic agents. Chemotherapy for OS often includes doxorubicin and cisplatin. Therefore, we explored combination treatment of OS cell lines and primary OS cell cultures with Ad5-Δ24RGD and doxorubicin or cisplatin. On OS cell lines, combination treatment was additive to synergistic. Surprisingly, however, on seven of eight primary OS samples no such combination effects were observed. In contrast, in many cases chemotherapy even inhibited CRAd-mediated cell killing. The inhibitory effect of doxorubicin on Ad5-Δ24RGD in primary OS cells appeared to correlate with slow cell growth rate; reduced viral replication and absence of chemotherapy-induced G2 cell cycle arrest. Our results point to the possibility that, at least for OS, virotherapy and chemotherapy should best not be performed simultaneously. In general, our work underscores the importance of testing new genetic anticancer agents and treatment regimens on primary cancer specimens.

Enhanced tumor cell kill by combined treatment with a small-molecule antagonist of mouse double minute 2 and adenoviruses encoding p53

Strategies to treat cancer by restoring p53 tumor suppressor functions are being actively investigated. These approaches range from expressing an exogenous p53 gene in p53 mutant cancers to antagonizing a p53 inhibitor in p53 wild-type (WT) cancer cells. In addition, exogenous p53 is used to strengthen the anticancer efficacy of oncolytic adenoviruses. Many cancers express high levels of the major negative regulator of p53, mouse double minute 2 (MDM2) protein. Recently, a novel class of highly potent and specific MDM2 antagonists, the Nutlins, was identified. We envisioned that Nutlins could protect both endogenous and exogenous p53 from MDM2-mediated inactivation. We therefore investigated treating human cancer cells with a combination of adenovirus-mediated p53 gene therapy and Nutlin. Combination treatment resulted in broadly effective cell kill of p53 WT and p53-negative cancer cells. Cytotoxicity was associated with profound cell cycle checkpoint activation and apoptosis induction. We also tested Nutlin in combination with oncolytic adenoviruses. Nutlin treatment accelerated viral progeny burst from oncolytic adenovirus-infected cancer cells and caused an estimated 10- to 1,000-fold augmented eradication of p53 WT cancer cells. These findings suggest that Nutlins are promising compounds to be combined with p53 gene therapy and oncolytic virotherapy for cancer. [Mol Cancer Ther 2007;6(5):1552–61]

Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site

Oncolytic viruses are promising anticancer agents because they selectively kill cancer cells and multiply within a tumor. Their oncolytic potency might be improved by expressing a therapeutic gene from the virus genome. In this regard, proper kinetics and level of transgene expression are important. In addition, expression of cytotoxic transgene products should be confined to cancer cells. Here, we developed oncolytic adenoviruses that provide transgene expression dependent on viral replication.

Intravenous administration of the conditionally replicative adenovirus Ad5-Δ24RGD induces regression of osteosarcoma lung metastases

Metastatic osteosarcoma (OS) has a very poor prognosis. New treatments are therefore wanted. The conditionally replicative adenovirus Ad5-Δ24RGD has shown promising anti-tumor effects on local cancers, including OS. The purpose of this study was to determine whether intravenous administration of Ad5-Δ24RGD could suppress growth of human OS lung metastases. Mice bearing SaOs-lm7 OS lung metastases were treated with Ad5-Δ24RGD at weeks 1, 2 and 3 or weeks 5, 6 and 7 after tumor cell injection. Virus treatment at weeks 1–3 did not cause a statistically significant effect on lung weight and total body weight. However, the number of macroscopic lung tumor nodules was reduced from a median of >158 in PBS-treated control mice to 58 in Ad5-Δ24RGD-treated mice (p = 0.15). Moreover, mice treated at weeks 5–7 showed a significantly reduced lung weight (decrease of tumor mass, p < 0.05), a significantly increased body weight gain (decrease of disease symptoms, p < 0.005) and a reduced number of macroscopic lung tumor nodules (median 60 versus > 149, p = 0.12) compared to PBS treated control animals. Adenovirus hexon expression was detected in lung tumor nodules at sacrifice three weeks after the last intravenous adenovirus administration, suggesting ongoing viral infection. These findings suggest that systemic administration of Ad5-Δ24RGD might be a promising new treatment strategy for metastatic osteosarcoma.

Replacement of Native Adenovirus Receptor-Binding Sites with a New Attachment Moiety Diminishes Hepatic Tropism and Enhances Bioavailability in Mice

The in vivo efficacy of adenoviral vectors (AdVs) in gene delivery strategies is hampered by the broad tissue tropism of the virus and its efficient binding to human erythrocytes. To circumvent these limitations, we developed a prototype AdV lacking native binding sites. We replaced the adenoviral fiber with a chimeric molecule consisting of the fiber tail domain, the reovirus sigma1 oligomerization domain, and a polyhistidine tag as model targeting moiety. We also abolished the integrin-binding motif in the penton base protein. The chimeric attachment molecule was efficiently incorporated onto AdV capsids, allowed efficient propagation of AdV without requirement for complementing fiber and conferred highly specific tropism to the AdV. Importantly, the targeted AdV exhibited markedly reduced tropism for liver cells. In comparison with control AdV with native tropism, the targeted AdV showed 1000-fold reduced transduction of HepG2 cells and 10,000-fold reduced transduction of mouse liver cells in freshly isolated liver slices. After intravenous inoculation of C57BL/6 mice, the targeted AdV exhibited delayed clearance in comparison with the native AdV, leaving approximately 10-fold greater levels in the blood 2 hr after inoculation. For all tissues analyzed, the targeted AdV displayed significantly reduced in vivo transduction in comparison with the native vector. Furthermore, in contrast to the native AdV, the targeted AdV did not bind human erythrocytes. Together, our findings suggest that the targeted AdV design described here provides a promising platform for systemic in vivo gene delivery.

Small cell osteosarcoma of a toe phalanx: a case report and review of literature

This report describes the radiological and histological findings of a small cell osteosarcoma of a toe phalanx in a 38 year old man. This man presented with pain, swelling and redness of the left third toe. Medical history revealed an osteomyelitis of this toe eight years prior. Based on clinical findings and medical history the lesion was diagnosed as an osteomyelitis. However, peroperatively the lesion had a malignant aspect. Histological examination revealed a small cell osteosarcoma of the proximal phalanx.Osteosarcoma of the foot and especially of the tubular bones is rare. Moreover small cell osteosarcoma is a rare subtype of osteosarcoma. This case demonstrates that medical history and clinical examination can be misleading. In patients with apparent bone destruction, a malignancy must always be excluded prior to treatment. It emphasises the care that should be taken in the process of formulating a diagnosis.