Thermosensitizers that alter the pH of the microenvironment of tumor cells (pHe) or alter the ability of tumor cells to regulate intracellular pH (pHi) may improve tumor control. However, regions of chronic hypoxia and acidosis, brought about by substrate limitation, exist not only in tumors but also in normal bone marrow; and bone marrow stem cells are exquisitely sensitive to hyperthermia. Therefore, the effects of sensitizers that modulate pHi on the thermal response of normal bone marrow must be assessed, since bone marrow may be a limiting normal tissue. Bone marrow contains numerous populations that primarily use glycolysis for energy production, and contains cells that are resistant to exposure to 20 mM lactate. These characteristics suggest that cells within marrow may be adapted to low pHe. Accordingly, it is hypothesized that bone marrow may contain stem cells that are adapted to low pHe and that these cells may be responsive to the hyperthermia sensitizers that act by inhibiting H+ exchange. Furthermore, the distribution of stem and progenitor cells in bone marrow will impose a differential response to hyperthermia and to the response of the thermal sensitizer that act by inhibiting proton exchange. The hypothesis will be tested by determining: 1) If elements of murine bone marrow are adapted to low pHe and if this adaptation is location specific; and 2) The extent to which amiloride (a clinically used diuretic which inhibits Na+/H+ antiport), quercetin (a bioflavinoid that inhibits the H+:lactate symport), DIDS ( a stilbene derivative that inactivates the Cl-/HCO3- exchanger) or hyperglycemia combined with quercetin can sensitize various marrow populations (i.e. CFU-S12, CFU-GM, CFU-E and CFU-F) to hyperthermia in situ or in vitro. Intracellular pH will be determined by the fluorescent dye BCECF. Tibial marrow will be heated in situ by waterbath immersion of the legs of anesthetized (pentobarbital) C57bl/6 mice, or heated in vitro by immersion of tissue culture tubes containing suspensions of nucleated marrow cells. CFU-S will be cultured as spleen colonies in lethally irradiated hosts. CFU-GM will be cultured in soft agar with recombinant CSF-GM as the source of colony stimulating factor, CFU-F will be grown in complete medium supplemented with fetal calf serum and CFU-E in 8% methylcellulose with erythropoietin. These studies will demonstrate that cells exist in normal marrow that are adapted to low pHe and that the addition of the thermosensitizers that alter pHi will preferentially sensitize these cells to hyperthermia. An understanding of the marrow micromilieu and stem cell distribution will improve our understanding of the response of this unique normal tissue to cytotoxic injury, and identify the possible mechanisms of injury imposed in a normal tissue by hyperthermia alone or in combination with agents that interfere with cellular proton control.
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