Sang Hie Kim

The Enhanced Killing of Irradiated HeLa Cells in Synchronous Culture by Hyperthermia

Mitotically synchronized cultures of HeLa S-3 cells were subjected to the treatment of radiation (400 rad), hyperthermia (43/sup 0/C), and a combination of both at different phases of the division cycle. The radioresistance was most pronounced in the mid G-1 and late S phases, while thermal resistance was greatest in the early G-1 phase and steadily decreased as cells entered the S phase. Cells in the late S and early G-2 phases were found to be most sensitive to hyperthermia. The sequential treatment of radiation immediately followed by hyperthermia resulted in an enhanced cell killing throughout the cell cycle with a marked synergism occurring in cells in the late S phase. The age-response function of the combined treatment was more similar to that of the thermal age response.

The potentiation of radiation response on murine tumor by fludarabine phosphate

Fludarabine phosphate is a synthetic analog of beta-arabinofuranosyl adenine (beta-ara-A), an anti-viral agent. Since beta-ara-A has been shown to be an effective inhibitor of potentially lethal damage (PLD) repair in cell culture system but ineffective in in vivo tumors, we carried out experiments to determine whether fludarabine phosphate which is not inactivated by adenosine deaminase potentiates the radiation effects on in vivo murine tumor. The combined effects of single acute fludarabine phosphate (600 mg/kg) and single dose of X-irradiation (20 Gy) on Meth-A fibrosarcomas in BALB/c mice produced more than 90% tumor control, while the radiation alone resulted in less than 10% tumor control. The radiosensitizing effect by fludarabine phosphate was higher when the drug was administered immediately prior to X-irradiation. The dose modifying factor of fludarabine phosphate is estimated to be 1.6 at 400 mg/kg. Experiments with fractionated irradiation and fludarabine phosphate similarly showed a high rate of tumor control. The present study suggests that inhibitors of PLD repair including several antiviral agents may have potential utility in the treatment of some radioresistant human tumors by radiotherapy.

Killing of Glucose-Deprived Hypoxic Cells with Moderate Hyperthermia'

Investigations were carried out to determine the importance of glucose as a modifying factor for hyperthermic cellular damage. HeLa S-3 cells were heated under oxic and hypoxic conditions, in the presence and absence of D-glucose. Temperatures as low as 41?C selectively enhanced killing of the glucose deprived hypoxic cell. Glucose deprivation (37?C, 2 hr) or heat alone (41?C for 2 hr in regular medium with glucose) produced minimal cell kill under oxic or hypoxic conditions. No enhancement was seen with hyperthermia under oxic condition in the absence of glucose. The result suggests that the interplay between glucose concentration is important for hyper

POTENTIATION OF RADIATION EFFECTS ON MULTICELLULAR TUMOR SPHEROIDS (MTS) OF HELA CELLS BY LONIDAMINE

Lonidamine is a potent inhibitor of spermatogenesis and a hyperthermic sensitizer. The previous study of lonidamine and radiation using two murine tumors demonstrated that tumor cure rates were significantly increased by radiation and concomitant lonidamine. In an effort to determine the radiobiologic factors involved with the potentiating effect of radiation by lonidamine, a series of cell culture studies were carried out using multicellular tumor spheroids (MTS) of HeLa Cells. When the MTS were treated with lonidamine in combination with fractionated irradiation, remarkable enhancement of growth inhibition was observed at the drug concentration of 10 micrograms/ml. On the other hand, there was no demonstrable enhancement of growth inhibition induced by a single dose of irradiation. Although the present findings would be consistent with the inhibitory action of potentially lethal damage repair of radiation by the drug, an alternative possibility is that the cells that have received the combined treatment have undergone a metabolic change, which has altered their sensitivity to the growth inhibitory effects of lonidamine. Based on the studies reported here and in mice, it is suggested that continued drug exposure over a prolonged period may provide an enhanced therapeutic effect, even in tumor varieties where the drug has no apparent antitumor activity on nonirradiated cells.

Radiosensitization of two murine fibrosarcomas with 6-thioguanine.

In Vivo murine tumor experiments were carried out to determine whether 6-thioguanine (6-TG) could enhance the cytotoxic effects of radiation on tumors. The combined effects of single and fractionated x-irradiation were evaluated on the transplanted methylcholanthrene induced fibrosarcoma (Meth-A) in BALB/c mice, a moderately radioresponsive tumor and on the radiation induced fibrosarcoma (RIF) in C3H/He mice, a highly radioresistant tumor. The combined treatment of single administration of 6-TG (25 mg/kg) and of x-irradiation (20 Gy) on Meth-A tumors produced more than 90% tumor control, whereas the radiation alone resulted in less than 5% tumor control. The radiosensitizing effect by 6-TG was higher when the drug was administered either 1 to 8 hr prior to or 24 hr after x-irradiation. The dose modification factor of single dose 6-TG (10 mg/kg) is estimated to be 1.47 for Meth-A tumor and 1.25 for RIF tumor. The tumor control rates of fractionated irradiation alone and with concomitant 6-TG in Meth-A tumors were 14% and 59%, respectively. Based on the studies reported here and well documented pharmacokinetics in humans, it is suggested that combined radiation therapy and 6-TG may provide an enhanced therapeutic effect even in tumor varieties where the drug has no apparent anti-tumor activity on non-irradiated cells.

A protein fraction (NHG) from serum of normal humans which is cytotoxic for HeLa cells in culture

A fraction of the alpha-globulins (NHG) from normal human serum was cytotoxic for mouse L-cells in culture and Meth A tumors in mice. NHG inhibited the growth in vitro of human colon cancer (HT-29), melanoma (RPMI 7931) and a neuroblastoma cell line. Survival of HeLa S-3 cell colonies after 24 h exposure to 25, 50, 75 or 100 micrograms NHG/ml medium was 86%, 77%, 40% and 10%, respectively. Whole human serum or purified serum albumin had no anti-HeLa cell activity. These results confirm the presence of a protein in human serum with antitumor activity. An assay for NHG using HeLa S-3 tumor cells is described.

Selective Killing of Glucose-Deprived Hypoxic Cells by Hyperthermia: I. Protection by Purine Ribonucleosides

Energy deprivation increases sensitivity to killing by hyperthermia. Hypoxic cells become dramatically sensitive to heat under glycolytic inhibition or glucose deprivation. To define the role of glucose metabolism in hypoxic cells in the presence or absence of elevated temperatures, cell culture studies were carried out to determine whether the enhanced cell killing of glucose-deprived hypoxic cells could be reversed by nucleoside supplementation. The results with HeLa cells showed that purine ribonucleosides were capable of reversing the enhanced heat-induced cytotoxicity under appropriate cultural conditions. Pyrimidine ribonucleosides and deoxyribonucleosides were ineffective. Based on the known metabolism of purine ribonucleosides, it is postulated that protection from hyperthermic killing by purine nucleosides comes about as a result of increased energy production via the purine nucleotide cycle.

Hypoxic cell radiosensitization by moderate hyperthermia and glucose deprivation

Cell culture studies were carried out to determine whether moderate hyperthermia reduces the oxygen enhancement ratio of cells under well-defined cultural conditions. Using asynchronously growing HeLa cells, the OER of cells with and without glucose was determined following exposure of cells to moderate hyperthermia, 40.5 degrees C for 1 hr, immediately after X irradiation. The OER of cells with 5 mM glucose was 3.2, whereas the OER of glucose-deprived cells was reduced to 2.0. The pH of the cell culture medium was kept at 7.4 throughout the experiments. The present finding may provide a clue toward further enhancing the radiosensitization of hypoxic cells by heat.

Interaction of Hyperthermia and Pentamidine in HeLa S-3 Cells

Pentamidine is similar to rhodamine-123 in chemical structure and state of electron charge, and rhodamine-123 was previously shown to be a hyperthermic sensitizer under appropriate cell culture conditions. The present experiments were carried out to determine whether pentamidine would potentiate hyperthermic cell killing and, if so, under what cultural conditions. Exposure of HeLa cells to pentamidine (80 microM) up to 4 h was not cytotoxic in culture medium in the presence or absence of glucose at 37 degrees C and pH 7.4. Cells in the glucose-deprived medium became progressively sensitive to killing as temperature and drug concentration were increased. On the other hand, there was a moderate level of enhanced cell killing in the glucose-fed medium at 42 degrees C. The enhanced effects of heat by the drug were most pronounced under alkaline pH of the culture medium. The cell kill under acidic pH was far less than that observed at neutral or alkaline pH; these effects may be a result of decreased cellular uptake of pentamidine. Together with our previous results on rhodamine-123 and glycolytic inhibitors, the present data with pentamidine are consistent with the concept that deprivation of cellular energy increases sensitivity to cell killing by heat.