Enhancement of camptothecin-induced cytotoxicity with UCN-01 in breast cancer cells: abrogation of S/G(2) arrest.

PURPOSE: To determine the ability of UCN-01 to abrogate the cell cycle arrest induced by camptothecin (CPT) in tumor cells that lack p53 function, and therefore enhance the cytotoxicity of CPT in these cells in relation to normal cells with wild-type p53. METHODS: The responses of MDA-MB-231 and GI 101A breast cancer cells were compared to those of normal bovine endothelial cells. Cytotoxicity was assessed by the MTT assay, and the resulting data were modeled using median-effect analysis. Inhibition of DNA synthesis was determined by loss of [(3)H]thymidine incorporation, and cell cycle status was determined by flow cytometric analysis of propidium-iodide-stained nuclei. RESULTS: UCN-01, a specific inhibitor of protein kinase C (PKC) presently in clinical trials, abrogated CPT-induced activation of S and G(2) checkpoints in human MDA-MB-231 and GI 101A breast carcinoma cells, both of which are mutants for the p53 gene. This abrogation occurred with the use of sublethal doses (100 nM) of UCN-01 and correlated with the enhancement of CPT-induced cytotoxicity. Median-effect analysis showed that synergistic cytotoxic interactions existed between CPT and UCN-01 against these tumor cells. In normal cells, however, abrogation of the S phase arrest caused accumulation in G(0)/G(1) phase, perhaps by the presence of wild-type p53 activity, with no change in CPT-induced cytotoxicity. CONCLUSION: We have shown previously that the cytotoxicity of CPT is correlated with cell cycle response in normal and tumor cells. Low doses of CPT arrest cells in the G(2)/M phase and inhibit DNA synthesis, but higher doses cause arrest of cells in S phase. Thus modulation of events at the S and G(2) checkpoints may provide an opportunity to enhance CPT-induced cytotoxicity in tumor cells. The results of this study indicate that UCN-01 enhances the progression of tumor cells through S phase thus greatly increasing CPT-induced cytotoxicity. Normal cells, however, are able to arrest in G(0)/G(1) and thus avoid the increased toxicity induced by CPT. Our findings suggest potential usefulness of combining UCN-01 in topoisomerase I inhibitor-based drug therapy for the treatment of breast cancer with a dysfunctional p53 gene.

Sensitivity to camptothecin of human breast carcinoma and normal endothelial cells.

PURPOSE: To assess parameters that might determine resistance to the topoisomerase I inhibitor, camptothecin (CPT), the sensitivities of three established human breast cancer cell lines (ER-) and of normal bovine endothelial cells to CPT in the free form and incorporated into liposomes (LCPT), were contrasted with topoisomerase I (topo I) content and activity, and with cell cycle response to CPT treatment. METHODS: Drug sensitivities were determined using the tetrazolium dye assay and by 3H-thymidine incorporation. Topo I levels were determined by Western blot analysis, and catalytic activity was determined with a plasmid relaxation assay, using nuclear protein from each cell line. CPT stabilization of cleavable complexes in nuclear extracts was determined using a labeled oligonucleotide with a specific topo I cleavage site. Cell cycle response to CPT was determined by flow cytometric analysis of propidium iodide-stained nuclei. RESULTS: CPT was extremely potent against MDA-MB-157 cells with an IC50 value of 7 nM compared with IC50 values of 150 nM for GI 101A and 250 nM for MDA-MB-231 cells. In contrast, CPT inhibited the incorporation of 3H-thymidine at very low doses in GI 101A and MDA-MB-231 cells with IC50 values of 9 nM and 5 nM, respectively; while MDA-MB-157 cells did not stop incorporating 3H-thymidine until very high doses (500 nM) of CPT were used. When incorporated into multilamellar liposomes (LCPT), CPT retained its potency, with IC50 values similar to that of the free drug. No correlation was found between CPT-induced cytotoxicity and any of the topo I parameters determined. Cell cycle analysis, however, showed an accumulation of cells in G2/M phase after 24 h treatment with low doses (5 nM) of CPT in only GI 101A and MDA-MB-231 cells with no arrest in normal endothelial or MDA-MB-157 cells. At higher doses (50 nM), however, a dramatic accumulation of cells in the S phase was observed in MDA-MB-157, MDA-MB-231 and GI 101A cells. In contrast, a G2/M phase block was seen with the normal bovine endothelial cells using the higher doses of CPT. CONCLUSIONS: The results suggest that cell cycle regulation plays an important role in determining the effect of CPT on malignant and normal cells. The possible mechanisms explaining the sensitivities of the two cellular compartments to the action of CPT are discussed.

Camptothecin exhibits selective cytotoxicity towards human breast carcinoma as compared to normal bovine endothelial cells in vitro.

We have reported earlier that camptothecin (CPT) incorporated into multilamelar liposomes of appropriate lipid composition displayed effective anti-tumor activity with minimal host toxicity in a nude mouse model xenographed with the human breast carcinoma Clouser nut 1. To investigate this observation further, we have determined the differential effects of CPT on the Clouser tumor cells as well as normal vascular (BVEC) endothelial cells in culture. We report here that Clouser cells are approximately 200-fold more sensitive to CPT (IC50 = 4.0 nM) than the normal endothelial cells (IC50 approximately 1 microM) as assayed by MTT; however, CPT demonstrates a potent anti-proliferative activity on both cell lines at low drug concentrations as measured by [3H]thymidine uptake. At higher concentrations (> 25.0 nM), however, the Clouser cells maintained a higher percentage of cells capable of incorporating [3H]thymidine. No significant differences in the levels of topoisomerase 1 protein and in vitro enzymatic activity were seen; although, the Clouser cells showed a 2-fold greater incidence of cleavable complex formation by CPT in vivo. Based on the data presented here, we propose that the selective cytotoxic activity of CPT towards tumor cells may be a function of the tumor cells' reduced ability to prevent cleavable complex formation. We also propose that the antitumor effect of CPT may be enhanced in vivo by its anti-proliferative effect on vascular endothelial cells which are normally solicited to promote tumor growth.

Self-measurement of blood pressure: assessment of equipment. Canadian Coalition for High Blood Pressure Prevention and Control.

Equipment for the self-measurement of blood pressure is readily available to consumers. These devices use one or more surrogate (indirect) measures of pressure to estimate systolic and diastolic blood pressure. Manual auscultatory devices using stethoscope and sphygmomanometer have been adapted for home use, but a variety of automated devices based on auscultation, oscillometry, and other techniques are available and may be more suitable for individuals who have limited vision, hearing or dexterity. Despite the existence of voluntary evaluation protocols and mandatory manufacturing standards, blood pressure readings from some automatic devices may not be accurate. Some devices are packaged with insufficient information to ensure proper use, and most individuals need some form of guidance in their use and calibration testing. If self-measurement of blood pressure is to be of benefit, the health care professional must recommend only those devices that are accurate and suitable to the patient or client. The Canadian Coalition for High Blood Pressure Prevention and Control will endeavour to develop a regular means by which health care professionals can keep informed of available devices for blood pressure self-measurement.

Host cellular annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts.

A significant amount of host cellular annexin II was found to be associated with human cytomegalovirus isolated from cultured human fibroblasts (approximately 1,160 molecules per virion). This composition was established by four different analytical approaches that included (i) Western blot (immunoblot) analysis of gradient-purified virions with a monoclonal antibody specific for annexin II, (ii) peptide mapping and sequence analysis of virus-associated proteins and proteins dissociated from virus following EDTA treatment, (iii) electron microscopic immunocytochemistry of gradient-purified virions, and (iv) labeling of virus-associated proteins by lactoperoxidase-catalyzed radioiodination. These results indicated that annexin II was primarily localized to the viral surface, where it bound in a divalent cation-dependent manner. In functional experiments, a rabbit antiserum raised against annexin II inhibited cytomegalovirus plaque formation in human foreskin fibroblast monolayers in a concentration-dependent manner. Cumulatively, these studies demonstrate an association of host annexin II with cytomegalovirus particles and provide evidence for the involvement of this cellular protein in virus infectivity.

Vascular endothelial growth factor increases urokinase receptor expression in vascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and endothelial cell-specific mitogen that stimulates urokinase-type plasminogen activator (uPA) activity in vascular endothelial cells. Here, we report that VEGF increases the high affinity binding of uPA to the same cells and that this binding is prevented by a peptide corresponding to the uPA receptor (uPAR) binding growth factor-like domain of uPA. Ligand cross-linking, ligand blotting, and uPA-Sepharose affinity chromatography revealed an increase in a cell surface uPA binding protein that corresponds to the uPAR on the basis of its affinity for uPA, M(r) of 50,000-55,000, and phosphatidylinositol-specific phospholipase C sensitivity. By Scatchard analysis, VEGF increased the number of uPAR molecules by 2.8-3.5-fold and concomitantly decreased their affinity for uPA. By northern blotting uPAR mRNA was increased in a dose- and time-dependent manner in response to VEGF. Taken together, these findings demonstrate that VEGF-induced angiogenesis is accompanied by increased uPAR expression and uPA activity on the endothelial cell surface. These observations are consistent with the notion that the uPA-uPAR interaction facilitates cellular invasion.

Human cytomegalovirus interaction with platelets and adhesive glycoproteins: significance in viral pathogenesis.

Human cytomegalovirus (HCMV) is released into plasma during active infection. To characterize cell-free virus interaction with blood components, the association of HCMV with washed platelets was assessed in binding assays. HCMV binding was regulated by divalent cations and inhibited in the presence of anti-HCMV antibody and excess unlabeled virus by 80% and 66%, respectively. Addition of plasma caused a 2.5-fold increase in binding to platelets. HCMV-induced platelet aggregation occurred more efficiently in plasma (14% +/- 4% in 5 min) than in buffer (8% +/- 2% in 15 min). Plasma fibronectin bound HCMV and increased the extent of HCMV-mediated aggregation of washed platelets by > 2-fold. Plaque assays indicated that platelet-associated HCMV retained its capacity to infect fibroblasts. Plasma and fibronectin inhibited viral infectivity by 81% and 70%, respectively. Thus, platelets and plasma components may play an intermediary role in HCMV infection and dissemination.

An endothelial cell-surface form of annexin II binds human cytomegalovirus.

Human cytomegalovirus was shown to bind to human umbilical vein endothelial cells in a specific, saturable and calcium-dependent manner (Kd = 7.9 pM (4 degrees C), 6469 virus binding sites/cell). Affinity adsorption of detergent-prepared lysates of surface-radiolabeled endothelial cells to virions resulted in the identification of cell-derived proteins of approximate M(r) 36,000 and 32,000 that bound cytomegalovirus. Protein sequencing of peptides obtained by cyanogen bromide cleavage demonstrated that the 36 kDa protein corresponded to human annexin II, and the 32 kDa protein was likely a degradation product. Purified annexin II was demonstrated to bind directly to virions (Kd = 57 nM, 688 annexin II binding sites/virion). These results provide evidence that an endothelial cell-surface form of annexin II acts as a receptor for cytomegalovirus, and indicate a previously undescribed role for annexin II.

In vitro angiogenic and proteolytic properties of bovine lymphatic endothelial cells.

The aim of the present study was to determine whether angiogenic cytokines, which induce neovascularization in the blood vascular system, might also be operative in the lymphatic system. In an assay of spontaneous in vitro angiogenesis, endothelial cells isolated from bovine lymphatic vessels retained their histotypic morphogenetic properties by forming capillary-like tubes. In a second assay, in which endothelial cells could be induced to invade a three-dimensional collagen gel within which they formed tube-like structures, lymphatic endothelial cells responded to basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in a manner similar to what has previously been observed with endothelial cells derived from the blood vascular system. Finally, since angiogenesis is believed to require extracellular proteolytic activity, we investigated the effects of bFGF and VEGF on lymphatic endothelial cell proteolytic properties by focussing on the plasminogen activator (PA) system. bFGF and VEGF increased urokinase, urokinase receptor, and tissue-type PA expression. This was accompanied by an increase in PA inhibitor-1, which is thought to play an important permissive role in angiogenesis by protecting the extracellular matrix against excessive proteolytic degradation. Taken together, these results demonstrate that with respect to in vitro morphogenetic and proteolytic properties, lymphatic endothelial cells respond to the previously described angiogenic factors, bFGF and VEGF, in a manner very similar to what has been described for endothelial cells derived from the blood vascular system.

Proficiency testing of factor VIII:C activity assays at the Canadian Red Cross Society National Reference Laboratory.

A national proficiency testing program has been established to monitor the accuracy and precision of factor VIII:C (FVIII:C) assays performed at the various Blood Services Centres of the Canadian Red Cross Society involved in the collection and processing of either donor blood designated for component production or plasma destined for fractionation. This paper describes the preparation, design, and results of the first four exercises involving 19 laboratories. The exercises were designed to allow the investigation of intralaboratory variability between replicate samples and precision of assays as well as the causes of interlaboratory variability. The implementation of this program has led to improved precision and interlaboratory agreement on FVIII:C assays. These improvements were achieved mainly as a result of a modification in the method for sample dilution, an increase in both replicate testing of the same sample and the number of sample dilutions, and a reduction in the number of reagent systems used by various centres.

Protein levels and plasmapheresis intensity.

In order to evaluate the influence of intensity of plasmapheresis on donor serum total protein and immunoglobulin concentrations, these parameters were measured monthly in two groups of plasma donors over a period of 6 months. Donors in group one donated 500-600 ml of plasma at weekly intervals and those in the other groups at intervals of 14 days or longer. Regular whole blood donors were used as a control group. The average concentrations of total protein and IgG immunoglobulin fraction in group one were significantly lower (P < 0.002) than those of the other two groups but they always remained well within the normal ranges. Although the mean total protein level of this group of donors fell significantly during the first 3 months, their values returned to almost baseline levels at the end of the study. No statistically significant difference from the initial concentrations was observed during monthly measurements of IgG, IgA and IgM levels among any of the groups studied. We conclude that removal of 500-600 ml of plasma at weekly intervals involves little, if any, risk of total protein or immunoglobulin depletion in donors who satisfy current criteria. This study also suggests that the frequency of IgG and IgM evaluations may be safely lowered and that IgA determinations may be limited to first time plasma donors.

Rapid immunoassays for the measurement of immunoglobulin G subclass concentration in immunoglobulin preparations and human serum.

Enzyme immunoassays (EIA) capable of determining total IgG1, IgG2, IgG3 and IgG4 subclass concentrations in human serum preparations have been developed. Subclass-specific monoclonal antibodies (mAbs) are bound to polyacrylamide bead-conjugated anti-mouse immunoglobulin antibodies. Bound immunoglobulins are detected with a peroxidase-conjugated anti-IgG antibody or a biotin-conjugated anti-IgG antibody followed by peroxidase streptavidin. The standard curves were found to be linear in the regions 16.0-2.0 micrograms/ml for IgG1, 4.0-0.5 micrograms/ml for IgG2, 0.4-0.06 micrograms/ml for IgG3 and 0.25-0.05 micrograms/ml for IgG4. Coefficient of variation (CV) values range from 0.32-7.32% for IgG1, 0.66-4.85% for IgG2, 1.62-6.85% for IgG3 and 0.05-6.47% for IgG4 standard curves. The inter-assay variability for the control human serum samples was 9.6% for IgG1, 6.7% for IgG2, 9.5% for IgG3 and 6.8% for IgG4.

Modulation of plasminogen activators and plasminogen activator inhibitors by TGF-beta, IL-1 alpha and EGF in fetal rat calvaria cells at different times of culture.

Fetal rat calvaria cells (RC cells) grown in long term culture in the presence of ascorbic acid and organic phosphate proliferate and differentiate to form mineralized nodules of bone. Since transforming growth factor beta (TGF-beta), interleukin 1-alpha (IL-1 alpha) and epidermal growth factor (EGF) affect both bone resorption and bone formation, we have studied the ability of these growth factors to affect plasminogen activators and plasminogen activator inhibitors release by RC cells at different times throughout this proliferation/differentiation sequence. Cultures in log phase growth (day 4), when first multilayering (day 7) and when bone nodules were forming (day 13) were exposed to either TGF-beta, IL-1 alpha, EGF or vehicle. Conditioned medium was collected after 6 and 24 h and plasminogen activators and plasminogen activator inhibitors were analysed by fibrin autography and reverse fibrin autography. TGF-beta-mediated changes in plasminogen activator were apparent at day 4. By day 7 two molecular weight species of plasminogen activator were noted; a 65 kDa species, prominent at 24 h exposure was blocked by anti-tPA antibody, and a 38 kDa plasminogen activator, prominent after 6 h of stimulation was not blocked by anti-tPA antibody. Plasminogen activator-plasminogen activator inhibitor complexes are also increased. IL-1 alpha caused similar increases in plasminogen activator and plasminogen activator inhibitor with maximal activity measured at day 13, coincident with the time when bone nodules were forming. EGF-mediated changes were less by comparison. TGF-beta significantly decreased bone nodule formation after both a 6 and 24 h serum-free exposure, whereas IL-1 alpha and EGF decreased nodule number only after the 24 h exposure. The data suggest that the three factors influence the expression of plasminogen activator and plasminogen activator inhibitor by RC cells and their effect is different at different times of culture.

The Canadian Red Cross plasmapheresis donor safety program: changes in plasma proteins after long-term plasmapheresis.

The Canadian Red Cross Blood Services have been harvesting plasma from whole blood by plasmapheresis procedure for the last 10 years. To date, we have performed approximately 230,000 procedures. To determine whether this procedure is a health hazard to an individual, a donor safety program was established in 1979 at the National Reference Laboratory. Serum levels of total protein, albumin, and immunoglobulins are monitored at intervals set by the Bureau of Biologics, Health and Welfare Canada. In this communication, we present a 10-year evaluation of this program. A comparison of the protein concentration distributions between first-time and long-term plasmapheresis donors showed no significant differences. Therefore, we have demonstrated that the donors are not at risk as the result of changes in the measured plasma protein levels following plasmapheresis procedure as performed over the last 10 years at The Canadian Red Cross Blood Services.

Biotinylated antibodies bound to streptavidin beads: a versatile solid matrix for immunoassays.

Streptavidin was covalently bound to commercially available polyacrylamide beads (3-10 microns diameter) by peptide bond formation between the carboxyl groups on the solid matrix and the amino groups of the soluble protein. Biotinylated antibody or lectin was linked to the polyacrylamide beads via the streptavidin molecules. Immunoassays for human IgA1, IgA2, IgE, and vitronectin were developed utilizing the antibody or lectin as a capture ligand. The protein being assayed was quantitated colorimetrically at 492 nm via horseradish peroxidase-conjugated antibodies.

An immunoassay for the determination of total IgA subclass antibodies in human serum.

An enzyme immunoassay capable of determining total IgA1 and IgA2 concentrations in human serum has been developed. Subclass-specific monoclonal antibodies are bound to polyacrylamide bead-conjugated anti-mouse immunoglobulin antibodies. Bound IgA is detected with an anti-IgA peroxidase conjugate and the standard curve is linear in the region 0.25 - 2.0 micrograms/ml. Coefficient of variation values range from 0.24 - 5.77% for the IgA1 standard curve and from 0.86 - 5.92% for the IgA2 standard curve. Inter-assay variation for the IgA1 and IgA2 control sample values were 8.2% and 13.4%, respectively.

Production of plasminogen activator and plasminogen activator inhibitor by bovine lymphatic endothelial cells: modulation by TNF-alpha.

We have investigated whether lymphatic endothelial cells in culture produce plasminogen activators (PAs) and their inhibitors (PAIs) and if these activities can be modulated by the inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). Examination by reverse fibrin autography of the conditioned medium from these cells revealed a PAI of Mr 50 kDa. Also evident by fibrin autography were two species of PAs, of Mr 110 kDa and Mr 60 kDa. The 110 kDa protein co-migrated with the PA-PAI complexes and the 60 kDa protein co-migrated with tissue Plasminogen Activator (tPA). Functional and immunological assays indicated the human TNF-alpha increased the type 1 plasminogen activator inhibitor (PAI-1) in a time dependent manner. Treatment of the cells with recombinant human TNF-alpha for 24 hours resulted in a 3 to 7 fold increase in the amount of PAI released into the conditioned media. Immunoblot analysis identified the PAI in the TNF-alpha treated cell conditioned media, as PAI-1. Deposition of PAI-1 in the extracellular matrix then became apparent. TNF-alpha increased 4 fold the amount of tPA-PAI-1 complexes (Mr 110 kDa) detected in the conditioned media. Free tPA (Mr 60 kDa) decreased to 1/5 of control. Net fibrinolytic activity, as determined by a chromogenic substrate assay, decreased after TNF-alpha treatment. No urokinase type Plasminogen Activator (uPA) activity was detected in control or treated cells. This fibrinolytic activity may be important in maintaining free fluid movement in the interstitium and lymphatic vessels and in inflammatory states this potential may be decreased by the increase in PAI-1.

A rapid procedure for the purification of IgA1 and IgA2 subclasses from normal human serum using protein G and jackfruit lectin (jacalin) affinity chromatography.

Immunoglobulin A (IgA) from pooled normal human sera was purified using antibody and protein G affinity chromatography and gel filtration high-pressure liquid chromatography (HPLC). This high purity product was separated into IgA1 and IgA2 subclasses utilizing the agarose-bound lectin 'jacalin'. Evaluation of product homogeneity by immunological testing confirmed greater than 95% purity. The total IgA1 and IgA2 recovered from sera was approximately 26% of the initial antibody present.