Activated human N-ras oncogene enhances x-irradiation repair of mammalian cells in vitro less effectively at low dose rate. Implications for increased therapeutic ratio of low dose rate irradiation

The effect of x-irradiation dose rate on the clonagenic survival of mouse embryo fibroblast cell line NIH/3T3 and its N-ras human oncogene transformed subline was studied. Both control and N-ras transformed cell lines were maintained in Dulbecco's modified Eagle's medium at 37 degrees C with 5% CO2. These cell lines were passaged twice weekly and the cells were irradiated in log phase on a 250 kVp orthovoltage unit at 5 or 200 rad/min, adjusting filtration and FSD to account for each dose rate. After irradiation, the cells were replated and colonies of greater than or equal to 50 cells were scored on day 7. D0 and n were calculated via linear regression analysis. There was a significant increase in saturation density and plating efficiency of N-ras transformed cells with loss of contact inhibition. There was no significant difference in radiosensitivity between the two cell lines at 5 rad/min. For NIH/3T3 D0 = 336, n = 2.19; for N-ras transformant D0 = 314, n = 2.35 (p = 0.65); however, irradiation at 200 rad/min revealed a significant survival advantage for the transformed line. For NIH/3T3 D0 = 145, n = 9.1 and for the N-ras transformed line D0 = 208, n = 4.05 (p = 0.0018). The data provide evidence that repair factors which govern irradiation survival may differ for high and low dose rate irradiation and that repair of high dose rate irradiation damage is enhanced directly or indirectly by expression of the N-ras oncogene. The data support hyperfractionated (low dose rate) irradiation for improving the therapeutic ratio during control of rapidly proliferating tumors expressing an activated N-ras oncogene.     

Tarsal navicular stress fractures: radiographic evaluation

Tarsal navicular stress fractures are a potential source of disabling foot pain in physically active individuals. The diagnosis of tarsal navicular stress fracture requires a high index of clinical and radiographic suspicion because the fracture is only rarely evident on routine radiographs or standard tomograms. The radiographic diagnosis of a tarsal navicular stress fracture may require anatomic anteroposterior tomograms or a radionuclide bone scan with plantar views. Radiographic examinations of 23 fractures in 21 patients are evaluated.     

Modeling stem cell population growth: incorporating terms for proliferative heterogeneity

Expansion of the undifferentiated stem cell phenotype is one of the most challenging aspects in stem cell research. Clinical protocols for stem cell therapeutics will require standardization of defined culture conditions. A first step in the development of predictable and reproducible, scalable bioreactor processes is the development of mathematical growth models. This paper provides practical models for describing cell growth in general, which are particularly well suited for examining stem cell populations. The nonexponential kinetics of stem cells derive from proliferative heterogeneity, which is biologically recognized as mitosis, quiescence, senescence, differentiation, or death. Here, we examined the assumptions of the Sherley model, which describes heterogeneous expansion in the absence of cell loss. We next incorporated terms into the model to account for A) cell loss or apoptosis and B) cell differentiation. We conclude that the basic assumptions of the model are valid and a high correlation between the modified equations and experimental data obtained using muscle-derived stem cells was observed. Finally, we demonstrate an improved estimation of the kinetic parameters. This study contributes to both the biological and mathematical understanding of stem cell dynamics. Further, it is expected that the models will prove useful in establishing standardization of cell culture conditions and scalable systems and will be required to develop clinical protocols for stem cell therapeutics.     

Synthesis and structure-activity relationships of 6,7-disubstituted 4-anilinoquinoline-3-carbonitriles. The design of an orally active,irreversible inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor-2 (HER-2)

A series of of 6,7-disubstituted-4-anilinoquinoline-3-carbonitrile derivatives that function as irreversible inhibitors of EGFR and HER-2 kinases have been prepared. These inhibitors have, at the 6-position, butynamide, crotonamide, and methacrylamide Michael acceptors bearing water-solublilizing substituents. These compounds were prepared by acylation of 6-amino-4-(arylamino)quinoline-3-carbonitriles with unsaturated acid chlorides or mixed anhydrides. We performed competitive reactivity studies showing that attaching a dialkylamino group onto the end of the Michael acceptor results in compounds with greater reactivity due to intramolecular catalysis of the Michael addition. This, along with improved water-solubility results in compounds with enhanced biological properties. We present molecular modeling results consistent with the proposed mechanism of inhibition. One compound, 5 (EKB-569), which shows excellent oral in vivo activity, was selected for further studies and is currently in phase I clinical trials for the treatment of cancer.     

Bee pollen-induced anaphylactic reaction in an unknowingly sensitized subject.

BACKGROUND: The food supplement bee pollen has been previously found to cause anaphylactic reactions. It has been proposed as useful for "everything from bronchitis to hemorrhoids." OBJECTIVE: This study describes an atopic patient who experienced a non-life-threatening anaphylactic reaction upon her initial ingestion of bee pollen. Microscopic examination of the pollen sample and ELISA inhibition assays were performed. RESULTS: The patient had a 7 mm/28 mm wheal/erythema reaction to bee pollen at 1 mg/mL concentration. Bee pollen caused 52% inhibition of IgE binding to short ragweed and 55% to ryegrass. Microscopic analysis revealed ragweed, Alternaria, Cladosporium, honeysuckle (Lonicera sp), privet shrub (Ligustrum sp), and vetch (Vicia sativa). CONCLUSIONS: An unknowingly sensitized atopic patient experienced an anaphylactic reaction after ingestion of a small quantity of bee pollen that contained pollens and fungi. Previously administered allergen immunotherapy that had reduced rhinitis symptoms did not prevent this allergic reaction.     

Modulation of redox signal transduction pathways in the treatment of cancer

Reactive oxygen species (ROS)-mediated damage to DNA is associated with induction of stress-activated protein kinases leading to secondary and tertiary effects on the nuclear matrix, cytoplasmic transport mechanisms, and altered mitochondrial and cell membranes. The cellular defenses against ROS damage are associated with up-regulation of gene products that can significantly alter cell biology, including antiapoptotic Bax family proteins and inflammatory proteins. Altered cell integrity can occur either directly or by indirect paracrine and juxtacrine interactions within tissues. Previous approaches toward therapeutic intervention against ROS damage have included administration of radical scavenger compounds, use of novel drugs that increase cellular production of constitutive antioxidants, or pharmacologic agents that modify the intracellular transport of antioxidants. Strategies to modify the cellular effects of ROS in hyperbaric oxygen injury to the lung, reperfusion injury to transplanted organs, and cancer have led to novel approaches of gene therapy in which the transgenes for antioxidant proteins can be expressed in specific tissues. Reducing tissue-damaging effects of ROS may have relevance to cancer patients by ameliorating normal tissue damage from ionizing irradiation therapy, photodynamic therapy, and cancer chemotherapy.     

Manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) administration protects mice from esophagitis associated with fractionated radiation

Intraesophageal administration of manganese superoxide dismutase-plasmid/liposome (MnSOD-PL) prior to single fraction radiation has been shown to protect mice from lethal esophagitis. In our study, C3H/HeNsd mice received fractionated radiation in two protocols: (i) 18 Gy daily for four days with MnSOD-PL administration 24 hr prior to the first and third fraction, or (ii) 12 Gy daily for six days with MnSOD-PL 24 hr prior to the first, third, and fifth fraction. Control radiated mice received either no liposomes only or LacZ (bacterial beta-galactosidase gene)-plasmid/liposome (LacZ-PL) by the same schedules. We measured thiol depletion and lipid peroxidation (LP) in whole esophagus and tested the effectiveness of a new plasmid, hemagglutinin (HA) epitope-tagged MnSOD (HA-MnSOD). In fractionation protocols, mice receiving MnSOD-PL, but not LacZ-PL (200 microl of plasmid/liposomes containing 200 microg of plasmid DNA), showed a significant reduction in morbidity, decreased weight loss, and improved survival. Four and seven days after 37 Gy single fraction radiation, the esophagus demonstrated a significant increase in peroxidized lipids and reduction in overall antioxidant levels, reduced thiols, and decreased glutathione (GSH). These reductions were modulated by MnSOD-PL administration. The HA-MnSOD plasmid product was detected in the basal layers of the esophageal epithelium 24 hr after administration and provided significant radiation protection compared to glutathione peroxidase-plasmid/liposome (GPX-PL), or liposomes containing MnSOD protein, vitamin E, co-enzyme Q10, or 21-aminosteroid. Thus, MnSOD-PL administration significantly improved tolerance to fractionated radiation and modulated radiation effects on levels of GSH and lipid peroxidation (LP). These studies provide further support for translation of MnSOD-PL treatment into human esophageal radiation protection.