Stockinette application over a non-prepped foot risks proximal contamination.

This study sought to determine if there was an increased risk for surgical site contamination during stockinette application for a lower extremity surgery draping technique. Utilizing a simulated, sterile surgical field, stockinettes were applied over 10 cadaver lower extremities that were contaminated with non-pathogenic Escherichia coli on the foot. Of those, five specimens were then disinfected with Chloroprep and another 5 did not undergo any disinfection. All the specimens in which the stockinette was applied over a non-prepped foot showed proximal contamination. No contamination occurred in any of the specimens where the foot was disinfected. Stockinette can be a source of surgical site contamination when placed over a non-prepared foot.

Creation of rabbit bone and soft tissue tumor using cultured VX2 cells.

BACKGROUND: To create rabbit VX2 bone tumors, it is surgically less demanding to implant VX2 cell suspensions than minced tumor fragments. A VX2 cell line that can be expanded using standard cell culture techniques might provide an unlimited supply of cells needed to create these bone tumors. Therefore, the aim of the present study was to establish a VX2 cell line and verify its tumorigenicity in an athymic mouse and rabbit animal model. MATERIALS AND METHODS: Minced VX2 tumor fragments were allowed to grow as a monolayer in 10 mL Dulbecco's modified Eagle medium/nutrient mixture F-12 (1:1) supplemented with 10% fetal calf serum and passaged multiple times. The tumorigenecity of the cultured VX2 cells were tested in athymic mice (intradermal tumor development) and in New Zealand white rabbits (bone and soft tissue tumor model). RESULTS: The VX2 cells proliferated rapidly in tissue culture flasks containing Dulbecco's modified Eagle medium/nutrient mixture F-12 medium supplemented with 10% fetal bovine serum. After reaching confluence, the VX2 cells can only be subcultured when plated at a greater density (e.g., at a dilution of 1:1). All 6 athymic mice developed tumors within 15 d of VX2 cell suspension implantation. In the rabbits, the VX2 cells were able to produce tumors in muscle tissue and in the distal femurs but not in the proximal tibia. CONCLUSIONS: VX2 cell lines can be successfully created from VX2 tumor fragments and passaged multiple times. In contrast to previous reports, the VX2 cells grown in vitro are capable of maintaining their tumorigenecity. However, successful tumor growth might depend on the initial number of cells implanted and the use of extracellular matrices for tumor proliferation.

Atorvastatin activates heme oxygenase-1 at the stress response elements.

Statins are known to inhibit growth of a number of cancer cells, but their mechanism of action is not well established. In this study, human prostate adenocarcinoma PC-3 and breast adenocarcinoma MCF-7 cell lines were used as models to investigate the mechanism of action of atorvastatin, one of the statins. Atorvastatin was found to induce apoptosis in PC-3 cells at a concentration of 1 µM, and in MCF-7 cells at 50 µM. Initial survey of possible pathway using various pathway-specific luciferase reporter assays showed that atorvastatin-activated antioxidant response element (ARE), suggesting oxidative stress pathway may play a role in atorvastatin-induced apoptosis in both cell lines. Among the antioxidant response genes, heme oxygenase-1 (HO-1) was significantly up-regulated by atorvastatin. Pre-incubation of the cells with geranylgeranyl pyrophosphate blocked atorvastatin-induced apoptosis, but not up-regulation of HO-1, suggesting that atorvastatin-induced apoptosis is dependent on GTPase activity and up-regulation of HO-1 gene is not. Six ARE-like elements (designated StRE1 [stress response element] through StRE6) are present in the HO-1 promoter. Atorvastatin was able to activate all of the elements. Because these StRE sites are present in clusters in HO-1 promoter, up-regulation of HO-1 by atorvastatin may involve multiple StRE sites. The role of HO-1 in atorvastatin-induced apoptosis in PC-3 and MCF-7 remains to be studied.

Evaluation of elution and mechanical properties of two injectable chemotherapeutic bone cements.

Chemotherapeutic bone cements can both stabilize the bone fractures as well as deliver chemotherapy agents directly to the bone metastatic site and adjacent soft tissue tumors. This study evaluated the in vitro elution and flexural properties of Vertebroplastic™ and Confidence Ultra™ bone cements (Depuy Spine Inc., Raynham, Mass., USA) containing methotrexate. In vitro elution was measured by placing bone cement specimens containing 4 different methotrexate amounts in 20 ml saline, and the methotrexate elution was measured at regular intervals for 672 h. The flexural properties of bone cement containing 2 different initial methotrexate amounts after storage in physiological saline were measured using a 3-point bending test. The drug elution rate depended on the initial methotrexate amount added and the type of bone cement used. The relationship between the initial drug amount added and the drug elution rate was not linear. Methotrexate elution decreased the flexural modulus and strength of specimens; this decrease was not proportional to the initial amount of methotrexate added. The results show that bone cements are well suited for use with chemotherapy agents. However, the elution and mechanical properties of each bone cement-drug amount combination should be thoroughly quantified in vitro before using such a combination in a clinical setting.

Brefeldin A activates CHOP promoter at the AARE, ERSE and AP-1 elements.

Brefeldin A induces apoptosis in PC-3 and MCF-7 cells at a concentration of 30 ng/ml. RT-PCR analyses showed up-regulation of CHOP/GADD153 and splicing of XBP-1 mRNA in brefeldin A-treated cells. CHOP promoter-luciferase reporter assays demonstrated activation of AARE, ERSE, and AP-1 elements of CHOP promoter by brefeldin A treatment. The activation of these elements was not affected by preincubation of cells with N-acetyl-cysteine (NAC), L: -buthionine-(S,R)-sulfoximine (BSO), and c-Jun N-terminal kinase (JNK) inhibitor (SP600125), suggesting that activation of CHOP promoter by brefeldin A may not involve oxidative stress or JNK signaling pathway. On the other hand, brefeldin A-induced apoptosis was not affected by NAC and BSO pretreatment, but was completely suppressed by JNK inhibitor pretreatment. Our results suggest that although CHOP is up-regulated by brefeldin A, it is not a major mediator of brefeldin A-induced apoptosis.

PTX1(ERGIC2)-VP22 fusion protein upregulates interferon-beta in prostate cancer cell line PC-3.

PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It is unrelated to the pituitary homeobox protein (Ptx1 or Pitx1), which regulates pituitary hormone gene expression, and its function is currently unknown. Recently, it was found to be a homolog of the yeast Erv41p, an endoplasmic reticulum (ER) resident protein involved in protein trafficking between ER and Golgi, and was renamed as ERGIC2. Ectopic expression of a partial sequence of PTX1 (Met84 - Leu225) as a VP22-fusion protein in prostate cancer cell line, PC-3, induced cellular senescence. Gene expression microarray analyses showed that interferon-beta (IFN-beta) and a number of IFN-inducible genes, among other genes, were upregulated by the PTX1-VP22 fusion protein. Upregulation of IFN-beta was confirmed by RTPCR and promoter-reporter assay. However, the upregulation of IFN-beta by the PTX1-VP22 fusion protein was not due to nuclear translocation of the PTX1 luminal domain.

Effects of PTX1 expression on growth and tumorigenicity of the prostate cancer cell line PC-3.

PTX1 is a gene identified by subtractive hybridization on the basis that it is expressed in normal prostate and not in prostate carcinoma. It encodes a nuclear protein that is downregulated in prostate carcinoma. Expression constructs containing PTX1 cDNA in both sense and antisense orientations were transfected into prostate tumor cell line, PC-3 cells. The effects of the expression of PTX1 and antisense PTX1 on PC-3 cells were examined using cell growth, proliferation, soft agar, invasion chamber, senescence-associated beta-galactosidase, and nude mice assays. Cells transfected with PTX1 construct in the sense orientation were growth-arrested. These cells displayed multiple morphological changes consistent with cellular senescence, including the expression of a senescence-associated beta-galactosidase. On the other hand, expression of antisense PTX1 RNA in PC-3 cells resulted in uncontrolled cell growth and increase of invasive potential. In nude mice, cells expressing antisense PTX1 grew sixfold faster than the control. These results suggest that PTX1 may play an important role in the growth and tumorigenicity of PC-3 cells.

Characterization of a variant of ERGIC2 transcript.

ERGIC2 (formerly known as PTX1) is a gene identified by subtractive hybridization on the basis that it is expressed in normal human prostate, but not in prostate carcinoma. It is unrelated to the gene encoding pituitary homeobox protein (Ptx1 or Pitx1), which regulates pituitary hormone gene expression. Based on sequence homology with the yeast Erv41 protein, it is suggested that the ERGIC2 protein is an endoplasmic reticulum (ER) resident protein involved in protein trafficking between the ER and Golgi intermediate compartment (ERGIC) and cis-Golgi. However, studies from our laboratory and others have shown that it may have other functions. In this study, we have identified a variant ERGIC2 transcript with a four base deletion at the junction of exons 8-9, resulting in frame shift after codon #189. As a result, a truncated protein of 215 residues (24.5 kDa) is predicted as compared with the 377-residue (42.6 kDa) wild-type (WT) protein. The truncated variant ERGIC2 protein loses 45% of the luminal domain and the transmembrane domain near the C-terminus, and this effectively abrogates its function as the ERGIC-Golgi protein transport shuttle. The variant, like the WT protein, was found to upregulate the heme oxygenase 1 gene, suggesting that it may be involved in the oxidative stress pathway.

Zinc Protoporphyrin Upregulates Heme Oxygenase-1 in PC-3 Cells via the Stress Response Pathway.

Zinc protoporphyrin IX (ZnPP), a naturally occurring molecule formed in iron deficiency or lead poisoning, is a potent competitive inhibitor of heme oxygenase-1 (HO-1). It also regulates expression of HO-1 at the transcriptional level. However, the effect of ZnPP on HO-1 expression is controversial. It was shown to induce HO-1 expression in some cells, but suppress it in others. The objective of this study is to investigate the effect of ZnPP on HO-1 expression in prostate cancer PC-3 cells. Incubation of PC-3 cells with 10 µ M ZnPP for 4 h showed only a slight induction of HO-1 mRNA and protein, but the induction was high after 16 h and was maintained through 48 h of incubation. Of all the known responsive elements in the HO-1 promoter, ZnPP activated mainly the stress response elements. Of the various protein kinase inhibitors and antioxidant tested, only Ro 31-8220 abrogated ZnPP-induced HO-1 expression, suggesting that activation of HO-1 gene by ZnPP may involve protein kinase C (PKC). The involvement of PKC α , ß , δ , η , θ , and ζ isoforms was ruled out by the use of specific inhibitors. The isoform of PKC involved and participation of other transcription factors remain to be studied.