Inhibition of PKA blocks fibroblast migration in response to growth factors.

Cell migration requires precise coordination of many signaling pathways to achieve directed motility. We report here that NIH3T3 fibroblasts expressing a dominant negative PKA subunit (dnPKA) show diminished migration in response to serum or growth factors. This effect is not a general effect on cell motility, but rather a decreased capacity to enhance migration in response to stimuli. Control (neo) and dnPKA cells show very similar haptotactic migration toward fibronectin, but dnPKA cells show reduced stimulation of migration in response to EGF/PDGF or serum. These effects were not due to alterations in cell growth or adhesion to fibronectin. Forskolin, which elevates cyclic adenosine monophosphate (cAMP) levels, dramatically inhibited neo cell motility in a scrape migration assay, although dnPKA cell migration was unaffected. The MEK selective inhibitor U0126 and the phosphatidyl-inositol-3 kinase (PI3K) inhibitor LY294002 inhibited migrating neo cells and were able to further inhibit residual dnPKA cell migration. Our data show that intermediate or well-controlled levels of PKA activity are required for optimal growth factor-stimulated migration in fibroblasts. PKA may play an important role in the signaling processes that lead to motility.

Selective drug resistant human osteosarcoma cell lines.

Chemotherapy in combination with surgery has been shown to be effective for the control of osteosarcoma. Development of resistance to chemotherapeutic agents is a recurring clinical problem. To investigate this phenomena, human osteosarcoma cells, TE-85, were exposed to increasing doses of Taxol or Taxotere during a 9-month period. Highly resistant subclones (TE-85TXL; TE-85TXR, respectively) were developed. Chemosensitivities are presented for TE-85 cell line and these new lines to Taxol, Taxotere, doxorubicin, cisplatin, and topotecan. Drug concentrations that inhibited cell growth by 50% compared with untreated cells were determined. The TE-85TXL cells showed resistance greater than 1,000-fold to Taxol and Taxotere and 60-fold to doxorubicin. The TE-85TXR cells showed resistance greater than 1,000-fold to Taxol, 800-fold to Taxotere, and 90-fold to doxorubicin. There was little cross resistance to topotecan and enhanced sensitivity to cisplatin. The role of P-170 glycoprotein in Taxol and Taxotere resistance was explored. Coincubation with verapamil, to block the actions of P-170 glycoprotein, partly reversed resistance to Taxol, Taxotere, and doxorubicin in both cell lines. Anti-P-170 glycoprotein antibodies revealed positive staining in TE-85TXL and TE-85TXR cell lines. Flow cytometry revealed reduced accumulation of doxorubicin in resistant cells. These data indicate that a human osteosarcoma cell line will develop resistance to Taxol and Taxotere, which is mediated in part by the P-170 glycoprotein.

Effect of ciprofloxacin on the proliferation of osteoblast-like MG-63 human osteosarcoma cells in vitro.

Locally applied antibiotic therapy is gaining popularity for the treatment of infections associated with open fractures and posttraumatic osteomyelitis. With use of local techniques, ciprofloxacin levels as high as 1,300 microg/ml, or over 200 times the bone levels achieved with intravenous administration, have been reported. To study the possible effects of ciprofloxacin on bone, osteoblast-like cells from the MG-63 human osteosarcoma cell line were studied. The cells were grown in antibiotic-free media and exposed to concentrations of ciprofloxacin at 0, 10, 100, 200, and 1,000 microg/ml to establish an initial dose-response curve. Media containing the appropriate dose of ciprofloxacin were changed every 24 hours. Cell number and [3H]thymidine incorporation per cell were determined at 0, 24, and 72 hours. A second dose-response curve was performed at concentrations of 0, 10, 20, 40, and 80 microg/ml. Three experiments, each with four observations, were performed. The results of this study demonstrated that ciprofloxacin caused significant decreases (p < 0.05) in cell number at 40 microg/ml at 24 hours and 20 microg/ml at 72 hours. [3H]thymidine incorporation per cell decreased significantly at levels of 80 microg/ml at 24 hours and 20 microg/ml at 72 hours. The authors conclude that reported local levels of ciprofloxacin seen in vivo inhibit the proliferation of human osteoblast-like cells in vitro.

Effect of cefazolin and vancomycin on osteoblasts in vitro.

The effect of cefazolin and vancomycin on osteoblast-like cells was studied. Cells from the MG-63 human osteosarcoma cell line were grown in antibiotic free media and exposed to concentrations of cefazolin and vancomycin at order of magnitude intervals between 0 and 10,000 microg/ml. For cefazolin, a second interval was performed between 100 and 1000 microg/ml to define toxic levels more accurately. Cell number and 3H-thymidine incorporation at 0, 24, and 72 hours were determined. The results of this study show that local levels of vancomycin of 1000 microg/ml and less have little or no effect on osteoblast replication, and concentrations of 10,000 microg/ml cause cell death. Concentrations of cefazolin of 100 microg/ml and less have little or no effect on osteoblast replication, 200 microg/ml significantly decrease cell replication, and 10,000 microg/ml cause cell death. The authors conclude that vancomycin is less toxic than is cefazolin to osteoblasts at higher concentrations and may be a better antibiotic for local administration in the treatment of similarly sensitive bacterial infections.

Bone toxicity of locally applied aminoglycosides.

The effect of tobramycin on osteoblasts was studied. Osteoblast-like cells from the MG-63 human osteosarcoma cell line were grown in antibiotic-free media and exposed to concentrations of tobramycin: first at order of magnitude intervals between 0 and 10,000 micrograms/ml, and then at closer intervals between 100 and 1,000 micrograms/ml to more accurately define toxic levels. Cell number and 3H-thymidine incorporation at 0, 24 and 72 h were determined. Similarly, primary cultures of rat osteoblasts were exposed to the same concentrations of tobramycin to confirm the validity of the MG-63 cell line as a model for nontransformed cells. The results of this study demonstrate that local levels of tobramycin of < or = 200 micrograms/ml have little or no effect on osteoblast replication. Concentrations of 400 micrograms/ml significantly decreased cell replication, and concentrations of 10,000 micrograms/ml caused cell death. Results obtained with primary rat osteoblast cultures were similar to those obtained from the MG-63 cultures at the tested tobramycin concentrations.