MECHANISMS OF TUMOR RADIORESISTANCE

The goals of our research program are to define molecular, biophysical and physiological factors that contribute to and predict for tumor radioresistance, and to develop novel strategies of radiosensitization that can be targeted to individual tumors. Current research projects include: the measurement of tumor hypoxia with novel radiodiagnostics and Eppendorf pO₂ microelectrodes; the identification of molecular and biophysical factors associated with the intrinsic radiosensitivity of tumor cells; the characterization of molecular lesions associated with single-hit inactivation; and, mechanistic studies of solid tumor response to photodynamic therapy (PDT).

Hypoxic tumor cells are 2.5 to 3.0 times more resistant to killing by ionizing radiation than are aerobic cells. The fraction of clonogenic cells within solid tumors that exhibit maximum radioresistance is defined as the tumor hypoxic fraction (HF). Recent microelectrode studies with human tumors of the cervix and the head and neck indicate that low oxygen levels in solid tumors strongly predict for treatment failure after radiation and other therapies. Hypoxic microenvironments in tumors were found also to promote mutation and metastases and to select for aggressive tumor phenotypes. A clinical technique that could accurately measure this tumor property, preferably by noninvasive procedures, would find broad application.

Our research has built upon our discovery that nitroaromatic compounds are selectively reduced within viable cells and become covalently bound to intracellular molecules at rates inversely proportional to oxygen concentration. The amount of such drug that becomes bound to tumor tissue was proposed as a positive marker of hypoxia and radioresistance. Several hypoxic markers of the iodinated azomycin-nucleoside class have been synthesized and tested in vitro and in animal tumors for their ability to measure tumor HF. Most of our synthetic chemistry effort over the past year focused on the manufacture of novel azomycinchelates to which Technetium (Tc)-99m could be linked. Over fifteen novel markers of this class have now been synthesized, radiolabeled and evaluated in our preclinical screening systems. When administered to severe combined immunodeficient (scid) mice intravenously, most of the compounds rapidly distribute to different organs according to compound lipophilicity and tissue blood flow. Tc-99m is then cleared from all tissues by an undefined process with a halflife of 10 to 12 hr. This is also the clearance rate of pertechnatate (TcO₄)- when it is administered intravenously to EMT-6 tumor-bearing scid mice. Recently, our research has identified a novel class of azomycin-chelates that are selectively reduced within hypoxic cells and retained at rates that mimic the radiobiology oxygen effect. Selected markers of this class have been labeled with Tc99m or Cu-67 and administered to EMT-6 tumor-bearing mice and rats with Dunning prostate carcinomas. Whole animal images acquired by planar nuclear medicine techniques indicated selective retention in tumors with 15 to 20% HF. The best marker of this class, designated FC-316, was found to label the hypoxic R3327-AT tumors with 3 to 5X the specific activity found in R3327-H (nonhypoxic) tumors. Additional analogues of these azomycin-chelates are being synthesized for structure/activity studies of this hypoxic marking reaction in vivo.

The effect of anesthesia on Eppendorf pO₂ electrode polarography was investigated. R3327-AT tumors of 10 to 15 cm³ volume were each subjected to electrode measurements of pO₂ along different tracks under conditions of animal restraint and four different anesthetic procedures. Median values of pO₂ obtained from tumors in animals that were anesthetized with Flurotec and breathing air, 100% O₂ or carbogen were 3.8 to 4.2 times higher than those obtained while animals were restrained and breathing air. When animals were deeply anesthetized with ketamine plus xylazine, median pO₂ values were 0.7 times those obtained from animals that were restrained and breathing air. These data demonstrate that microelectrode measurements of tumor oxygenation are strongly dependent upon the physiologic parameters associated with the anesthesia employed. As yet, we have not determined whether these large changes in microelectrode pO₂ measurements result from blood flow and tissue perfusion changes in the animal tissues induced by the anesthetics or if the anesthetics themselves interfere with the polarographic assessment of oxygen. These data demonstrate that microelectrode validation measurements of tumor oxygenation must be performed on similarly treated animals, whether awake or anesthetized.

Custom-made Eppendorf pO₂ microelectrodes of 12 to 14 cm length were purchased for oxygen measurements in human prostate cancers. Prior to isotope implacement for brachytherapy, microelectrode measurements of prostate cancer oxygenation were acquired with ultrasound guidance. These studies showed that oxygen levels in prostate cancer tissue are usually lower than those in adjacent muscle, that the median pO₂ values obtained from individual tumors of similar pathologic grade are heterogeneous, that median pO₂ decreases with increasing tumor stage, and that microelectrode measurements can be influenced by the anesthetic employed.

The single-hit inactivation mechanism (a) described by the linear-quadratic equation is responsible for the majority of cell killing at low radiation doses and at 1.8 to 2.0 Gy, the most common dose fraction size used in cancer radiotherapy. Our research with synchronized human tumor cells showed that the large differences in radiosensitivity during interphase are eliminated at mitosis when intrinsic radiosensitivities become maximal and almost equal. These data suggested that chromatin in compacted form may be the cellular target for single-hit inactivation. Additional studies were performed with synchronized CHO-K1 (wild type) and CHO-xrs5 (mutant cell line defective in Ku-86) cells. Chromatin densities in mitotic and G1-phase cells were quantified by transmission electron microscopy and correlated with cellular radiosensitivity. For cells in mitosis, single-hit inactivation varied between 2.0 to 5.0 Gy^-1 and correlated with chromosome density. The DNA in G1-phase CHO-K1 cells was uniformly dispersed throughout their nuclei (excluding the nucleosome) while a significant fraction of DNA in G1-phase CHOxrs5 cells remained in condensed form associated with the nuclear membrane. These data suggest that DNA-protein kinase (of which Ku-86 is a functional component) may play a role in the processing of DNA at mitosis and that some component of their radiation hypersensitivity results from their DNA remaining in compacted form. These studies have suggested a novel mechanism for understanding mammalian cell inactivation by single-hit and multiple-hit mechanisms.

An expression vector which contained functional human Ku-86 cDNA under the control of a CMV promoter adjacent to puromycin resistance and green fluorescent protein (GFP) genes under a RSV promoter was constructed and transfected into CHO-xrs5 and CHO-K1 cells with the aid of lipofectamine. Ten transfected cell lines were selected by GFP expression and by resistance to puromycin. Ku-86 expression and radiosensitivity are being characterized. These studies will determine if reversion to radiation resistant phenotype by transfection with functional Ku-86 correlates with the improved dispersion of chromatin in interphase cells.

Our current research is investigating the molecular basis of radiation hypersensitivity of compacted chromatin. Alkaline and neutral comet assays of DNA damage indicate that the chromatin in mitotic chromosomes contains both single-- and double-strand DNA breaks which are rapidly repaired after cytokinesis. Compacted DNA may be hypersensitive to radiation if these normal DNA sublesions can interact with radiationinduced damage to produce lethal lesions. As well, compacted DNA may provide a more efficient target for electron track-end (high-LET) damages than dispersed DNA. Compounds that inhibit histone kinases and phosphatases and can modulate normal DNA condensation processes in cells are being investigated as radioprotective and radiosensitizing agents.

Our previous research showed that low energy electrons scattered from highZ materials resulted in much higher radiation doses at distances up to 0.1 mm from the scatterer. The increase in average absorbed dose to the bulk of 1% solutions of gold particles (1.5 to 3.0 mm diameter) was measured by Fricke dosimetry and cell killing. The dose increase was ~40% by both the chemical and biological assays. When gold particle solution was injected directly into EMT-6 tumors growing in scid mice prior to their irradiation by 8 Gy of Cs-137 g-rays, tumor cell killing by in vivo/in vitro assays was increased. These data demonstrate that if high-Z particles of appropriate size and at high enough concentration can be selectively delivered to solid tumors, the dose delivered to tumor tissue and the therapeutic ratio can be increased.

Biological characterizations of tumor PDT with Tolyporphin are continuing. Its relatively uniform biodistribution to most tissues of animals including EMT-6 tumors has been confirmed. Current studies are determining whether it is cleared from normal tissues more quickly than from tumors. Such an effect would produce a favorable drug distribution at later times for tumor illumination. As well, in vivo/in vitro assays of tumor cell viability will determine whether its PDT effect on tumors results from the direct killing of tumor cells or by secondary tumor cell death after destruction of tumor vasculature.

CHAPMAN, J.D., STOBBE, C.C., GALES, T., DAS, I.J., ZELLMER, D.L. BIADE, S., MATSUMOTO, Y. Condensed chromatin and cell inactivation by single-hit kinetics. Radiat. Res. 151:433-441, 1999.

DAS, I.J., CHAPMAN, J.D., VERHAEGEN, F.,ZELLMER, D.E. Interface Dosimetry in Kilovoltage Photon Beams. In Kilovoltage X-ray Beam Dosimetry for Radiotherapy and Radiobiology, edited by C.-M.C. Ma, J.Seuntjens. Madison Medical Physics Publ., pp. 239-260, 1998.

IYER, R., ENGELHARDT, E.L., STOBBE, C.C., SCHNEIDER, R.F., CHAPMAN, J.D. Preclinical assessment of hypoxic marker specificity and sensitivity. Int. J. Radiat. Oncol. Biol. Phys. 42:741-745, 1998.

MOVSAS, B., CHAPMAN, J.D., HORWITZ, E.M., PINOVER, W.H., GREENBERG, R.E., HANLON, A.L., IYER, R., HANKS, G.E. Hypoxic microregions exist in human prostate carcinoma. J. Urol. 53:11-18, 1999.

IYER, R., KIM, E., SCHNEIDER, R.F., CHAPMAN, J.D. A dual hypoxic marker technique for measuring oxygenation change within individual rodent tumors. Br. J. Cancer 78:163-169, 1998.

MORLIÈRE, P., MAZIERE, J.-C., SANTUS, R., SMITH, C.D., PRINSEP, M.R., STOBBE, C.C., FENNING, M.C., GOLBERG, J.L., CHAPMAN, J.D. Tolyporphin: A natural product from cyanobacteria with potent photosensitizing activity against tumor cells in vitro and in vivo. Cancer Res. 58:3571-3578, 1998.

ZELLMER, D.L., CHAPMAN, J.D., STOBBE, C.C., XU, F., DAS, I.J. Radiation fields backscattered from material interfaces: I. Biological effectiveness. Radiat. Res. 150:406-415, 1998.

^§   Fox Chase researcher

^a   R. Iyer: Present address--Lincoln Medical Center, Bronx, NY 10451-5589

^b   S. Gatt: Hebrew University, Hadassah School of Medicine, Jerusalem, Israel 91-010

^c   R. Santus: Museum National D'Histoire Naturelle, INSERM W.312, 75231 Paris, France

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998