Glenn C. Rice

Preferential radiosensitization of G1 checkpoint-deficient cells by methylxanthines

PURPOSEnTo develop a checkpoint-based strategy for preferential radiosensitization of human tumors with deficient and/or mutant p53.nnnMETHODS AND MATERIALSnA549 human lung adenocarcinoma cell lines differing in their expression of the p53 tumor suppressor gene were produced by transduction with the E6 oncogene from human papilloma virus type 16. The cells expressing E6 (E6+) lack a G1 arrest in response to ionizing radiation, are deficient in p53 and p21 expression, and exhibit a fivefold greater clonogenic survival following 10 Gy radiation.nnnRESULTSnPostirradiation incubation with millimolar concentrations of the methylxanthine pentoxifylline (PTX) results in preferential radiosensitization of the E6+ cells compared to the LXSN+ vector transduced controls. There is a threefold sensitization of the LXSN+ cells and a 15-fold sensitization of the E6+ cells, which results in equal clonogenic survival of the two lines. Flow cytometry reveals PTX abrogation of the radiation induced G2 arrest for both cell lines. PTX also prolongs G1 transit for both cell lines. Preliminary results are presented using a novel methylxanthine, lisofylline (LSF), which has similar cell cycle effects on G1 and G2 and achieves differential radiosensitization at micromolar concentrations that are sustainable in humans.nnnCONCLUSIONnThis checkpoint-based strategy is a promising approach for achieving preferential radiosensitization of p53- tumors relative to p53+ normal tissues.

Cell killing, radiosensitization and cell cycle redistribution induced by chronic hypoxia☆

Some of the biological changes associated with extreme hypoxia at 37 degrees C (less than 10 ppm pO2) were examined in Chinese hamster V79 cells. Specifically, extreme hypoxia caused an initial decrease in plating efficiency to 55% in 4 hr after the onset of hypoxia. Beyond this time, the decline in plating efficiency was more gradual reaching 35% of control at 20 hr. Flow microfluorimetry (FMF) studies, in which cells are sorted on the basis of DNA content and then assayed for viability, demonstrated that mid S phase cells were most sensitive to chronic hypoxia, with surviving fraction equal to 2.5% at 20 hr. Furthermore, the viability of G1 and G2/M cells, after 20 hr of hypoxic storage, was also reduced to 20 and 7.6%, respectively. Hypoxia also caused alterations in the cell cycle distribution of initially asynchronous cells, as determined by dual parameter FMF measurements of both cellular DNA content and incorporated BudR. In particular, G2/M cells completed mitosis, while G1 cells showed little or no movement. Lastly, cells stored in chronic hypoxia displayed an enhanced radiosensitivity when compared to acutely hypoxic cells. Possible reasons for these observations are discussed.

Modulation of adriamycin transport by hyperthermia as measured by fluorescence-activated cell sorting

SummaryHeat-induced (45.5°C) modification of adriamycin uptake and efflux were measured by flow cytometry in CHO cells in vitro. Administration of adriamycin with simultaneous 15-min or 30-min heat treatment increased drug uptake in a dose-dependent manner. Fluorescence-activated cell sorting showed that cytotoxicity to adriamycin was correlated with relative cellular concentration (fluorescence) for both unheated cells and those heated and simultaneously treated with adriamycin. However, if adriamycin administration followed the heat treatment, accumulation was significantly reduced, primarily as a result of decreased passive drug diffusion (rather than increased efflux) in the heated cells. Cells made heat-tolerant by prior heating also exhibited reduced adriamycin uptake 12 h later, and further heating did not increase uptake. Cell sorting experiments indicated that cytotoxicity of adriamycin was not necessarily correlated with intracellular drug levels when drug administration followed the heat treatment. Also, heat-sterilized cells exhibited a two-fold increase in adriamycin uptake over surviving cells, as assessed by simultaneous measurement of dansyl lysine and adriamycin content. These results indicate that sensitization to adriamycin by simultaneous heat treatment is probably due to increased drug uptake. The decreased sensitization observed when drug administration is followed by heating is probably the result of both decreased uptake and decreased drug DNA accessibility.

Use of N-σ-dansyl-L-lysine and flow cytometry to identify heat-killed mammalian cells

We have employed the nontoxic fluorescent membrane probe, N-σ-dansyl-L-lysine (DL) to study the effect of mild (45.5°C) heat shock on a variety of mammalian cell lines. It has been previously proposed by Humphries and Lovejoy (1983) that DL selectively partitions into (and diffuses through) membranes whose component molecules have undergone lateral phase separation resulting in the formation of phospholipid domains. Excellent flow cytometric resolution of the DL staining cells from several cell lines was obtained by using bivariate (forward angle light scatter versus DL-fluorescence) analysis. Dye uptake and release data as well as measurement of the octanol: water partition coefficient (7.2) all indicated that the stain was likely associated with the plasma membrane. After heating, all cell lines exhibited a time-dependent increase in the fraction of cells stained by DL. Nearly all of the DL-staining cells were propidium iodide and trypan blue excluding. Exclusion of erythrosin B or inclusion of fluoresc...

Detection of S-phase overreplication following chronic hypoxia using a monoclonal anti-BrdUrd

We have examined cytokinetic perturbations induced in Chinese hamster V-79 cells in vitro during and following exposure to chronic hypoxia employing simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content. These data indicate hypoxia inhibited G1 progression into S-phase, but did not significantly delay G2M division and progression into G1. Also, upon reaeration after 20 hr in hypoxia, cells originally in G1 exhibited significant kinetic delay. BrdUrd pulse/chase and pulse/fix data indicated DNA replication was reduced, but not completely inhibited during hypoxia. Also, between 6 and 20 hr of chronic hypoxia and following reaeration, a subset of the original S-phase cells overreplicated their DNA, such that these cells had greater than 2C DNA content. This subpopulation was estimated on the average to comprise approximately 20% of the total population (30% of the treated S-phase subpopulation) by 24 hr following reaeration after 20 hr hypoxia. These results are discussed in light of the similarities between overreplication and gene amplification observed under certain conditions with other agents, which like chronic hypoxia, are used to transiently disrupt DNA synthesis.

Correlation of mammalian cell killing by heat shock to intramembranous particle aggregation and lateral phase separation using fluorescence-activated cell sorting.

Heat shock induces a dose-dependent increase in the fraction of Chinese hamster ovary cells that stain the fluorescent membrane probe N-epsilon-dansyl-L-lysine (DL). Dansyl lysine has previously been shown to select for cholesterol-free membrane domains in phospholipid liposomes. We found that the fraction of cells excluding DL could be closely correlated to cell survival as assayed by 37 degrees C incubation following heat treatment. Fluorescence-activated cell sorting indicated that essentially all of the DL-staining cells were nonviable. Freeze fracture electron microscopy of sorted cells showed that all the cells that stained with DL also had highly suggested intramembranous particle (IMP) aggregation while DL-excluding cells did not. Furthermore, IMP aggregation was shown to occur immediately after heat shock and to precede DL staining. Treatment with other membrane-active agents such as ethanol, amphotericin B, filipin, procaine, and lidocaine (i) induced DL staining that was closely correlated to survival, (ii) induced dramatic cytotoxic sensitization when combined with heat, and (iii) induced aggregated IMPs at relevant cytotoxic concentrations. Several nonmembrane-active agents were examined; none induced DL staining, dramatic cytotoxic sensitization, or IMP aggregation. These results raise the possibility that heat shock inactivates mammalian cells primarily via nonspecific aggregation and denaturation of membrane proteins resulting in a lateral phase separation of membrane components, including the generation of phospholipid domains.

A predictive assay for human tumor cellular response to hyperthermia using dansyl lysine staining and flow cytometry

The heat response of five human tumor biopsies has been examined using the fluorescent probe dansyl lysine and multiparameter flow cytometry. Dansyl lysine has previously been shown to possess specificity for heat killed mammalian cells. The human tumors tested included a cervical squamous cell carcinoma, malignant melanoma, colon adenocarcinoma, ovarian carcinoma, and a mesothelioma. The samples were excised, mechanically disrupted into single cell suspensions and heated in vitro for various lengths of time at 45 degrees C. The cells were returned to 37 degrees C incubation for 12 to 15 hours prior to staining with dansyl lysine. The fraction of cells staining dansyl lysine was quantitated by flow cytometry after gating on high forward angle light scatter and 90 degrees C light scatter. This gate excluded much of the normal cell contamination within the tumor sample. The data show that the heat response of human tumor biopsies varied significantly, with cervical carcinoma and malignant melanoma being the most resistant and the mesothelioma and ovarian carcinoma the most heat sensitive. Finally, evidence is presented for the expression of thermotolerance in ovarian carcinoma and mesothelioma biopsies pre-heated in vitro. Dansyl lysine appears to be useful in measuring the intrinsic cellular heat sensitivity of human tumors and in determining the kinetics of decay of thermotolerance following an initial heat exposure.

Predictive Assays for Tumor Response to Single and Multiple Fractions of Hyperthermia

Local regional hyperthermia is currently being tested clinically in combination with radiation therapy or chemotherapy. The results of several clinical trials show that for superficial tumors, hyperthermia used in conjunction with radiotherapy improves the response rate over radiotherapy alone (Overgaard 1985).

Heterogeneity of heat response in murine, canine and human tumors: Influence on predictive assays

The heterogeneity of response to hyperthermia of cells taken from different regions of tumors was tested in a model tumor system (RIF-1) in the mouse and in specimens from spontaneous tumors taken from dogs and humans at the time of surgical resection. Cell survival was assayed by clonogenic survival in the murine tumor and by dansyl lysine staining in tumors from all three species. Using survival as an endpoint, it was found that the extent of heterogeneity depended on the temperature to which the tumor was heated and the duration of exposure. By increasing either of these factors, the coefficient of variation was increased. The large heterogeneity seen after in vivo heating could not be explained entirely by inhomogeneous heating within the tumor as evidenced by temperature mapping. It is concluded that other microenvironmental factors such as blood flow, pH, O2, and nutrient supply may cause variations in the heat response of the tumor cells in vivo. Little, if any, evidence of cellular heterogeneity was evident for all three species when comparisons were made between samples of 100-200 mg. The canine and human tumors were considerably more heat resistant when dansyl lysine was used as an endpoint. In the RIF-1 tumors, heterogeneity of heat response was greater after in vitro heating than after in vivo heating when small biopsy samples (10-20 mg) were taken, suggesting that some cellular heterogeneity was present.

THE USE OF DANSYL LYSINE TO ASSESS HEAT DAMAGE AND THERMOTOLERANCE OF NORMAL TISSUES

The fraction of cells excluding the fluorescent dye dansyl lysine has previously been shown to correlate well with heat-induced cell killing in a variety of mammalian cell lines and in murine tumors in vivo. Here we evaluate the usefulness of dansyl lysine as a probe for assessment of thermal damage and for measuring the kinetics of thermotolerance development and decay in murine normal tissues. Skin cells were heated in vivo with an initial treatment of 44 degrees C for 20 min by local radiofrequency. Bone marrow cells were heated at 42.5 degrees C for 20 min by whole body water bath immersion. Cell suspensions were prepared, heated in vitro for various lengths of time at 44 degrees C (skin) or 43 degrees C (bone marrow), and scored for the fraction of dansyl lysine-excluding cells. Skin and bone marrow cells expressed maximum thermotolerance by 8 and 6 hr, respectively and returned to normal heat sensitivity by 48 and 146 hr, respectively. The assay was not useful with skeletal muscle and liver, as we were not successful in obtaining viable, dansyl lysine-excluding cells from these tissues. Also, in our hands red blood cells, normal human leukocytes, mouse spleen and thymus cells all failed to stain dansyl lysine even after extreme heating. Dansyl lysine staining, particularly when combined with flow cytometry analysis, has been shown to be a useful method for assessing thermal damage and thermotolerance relatively rapidly in all tumor systems tested to date, and, as shown here, may possess utility in measuring similar endpoints for certain nonclonogenic normal tissues.