Oncogenic Basis of Radiation Resistance

ONCOGENIC BASIS OF RADIATION R ESISTANCE Usha Kasid,' Kathleen Pirollo,t Anatoly Dritschilo,* and Esther Changt-* 'Department of Radiation Medicine, Lombardi Cancer Center, Georgetown University, Washington, D.C. 20007; and Departments of *Pathology and *Surgery, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

I. Introduction 11. Radiation Response Phenotype A. Relative Radioresistance and Radiosensitivity: Radiation Survival Parameters B. Clonal Nature of the Radiation Response 111. Human and Rodent Cell Model Systems A. Radioresistant Human Squamous Cell Carcinomas B. Skin Fibroblasts from a Cancer-Prone Family C. NIH13T3 Transfectants D. Other Cell Lines IV. Mitogenic Signals and Radiation Response V. Transformation and Radiation Resistance VI. Radiation-Resistant Phenotype: Cause or Effect A. Multifactorial Nature of Radiation Response: Differential Gene Expression B. Molecular Targets of Ionizing Radiation VII. DNA Damage and Repair Cascade: Biochemical and Cellular Factors VIII. Modulation of Radiation Resistance: Therapeutic Implications of Oncogene Strategy IX. Conclusion References

1. Introduction

Evidence continues to accumulate implicating oncogenes in the development of neoplasia. T h e normal counterparts of these genes (protooncogenes) are involved in numerous vital cellular functions. The products of many of these protooncogenes have been shown to interact with one another as components of a signal transduction pathway that involves transmission of molecular/biochemical signals from the membrane to the nucleus directing the cells to divide or to differentiate (Weinstein, 1988a; Nigg, 1990; Bishop, 1991; Cantley et al., 1991). Ionizing radiation induces multiple cellular and biological effects either by direct interaction with DNA or through the formation of free 195 ADVANCES IN CANCER RESEARCH. VOL. 61

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radical species leading to DNA damage (Painter, 1980). These effects include cell cycle-specific growth arrest, repair of DNA damage, radicalscavenging proteins, gene mutations, malignant transformation, and cell killing. The mechanisms underlying sensitivity or resistance of certain mammalian cells to the toxic effects of y-radiation have been a topic of a number of studies (Taylor et al., 1975; Paterson et al., 1976; Weichselbaum et al., 1977; Arlett and Harcourt, 1980; Cox and Masson, 1980; Little et al., 1989; Little and Nove, 1990). T h e individual molecular events and specific genes involved in the resistance to y-radiation will affect both normal cellular protection from radiation damage and the failure of tumors to respond to radiation therapy. In recent years, several lines of investigation have coalesced to demonstrate a link between certain oncogenes and the phenomenon of cellular resistance to ionizing radiation. T h e raf-l l protooncogene has been associated with radiation-resistant human laryngeal squamous carcinoma-derived cells (Kasid el al., 1987a, 1989a),as well as radiation-resistant noncancerous skin fibroblasts from a specific cancer-prone family with Li-Fraumeni syndrome (Chang et al., 1987; Pirollo et al., 1989). Transfections not only of the ruf- 1 oncogene, but also of other protein-serine kinase oncogenes, mos and cot, have been shown to confer the radiationresistant phenotype on the recipient cells (Pirollo et al., 1989; Suzuki et al., 1992). An increase in level of radiation resistance was also demonstrated by transfection of Ha-, Ki-, or N-ras oncogene into murine hematopoietic cells or NIH/3T3 cells (FitzCerald et al., 1985; Sklar, 1988), and a synergistic increase in the level of radiation resistance of primary rat embryo cells was seen by cotransfection of ras and m y oncogenes (Ling and Endlich, 1989; McKenna et al., 1990b). Therefore, there is a growing body of evidence indicating that oncogenes play a major role in cellular resistance to ionizing radiation. Here we will review some of that evidence and attempt to formulate the themes underlying the oncogenic basis of radiation resistance. II. Radiation Response Phenotype

A. RELATIVE RADIORESISTANCE AND RADIOSENSITIVITY: RADIATION SURVIVAL PARAMETERS T h e central hypothesis concerning the outcome of the y-irradiation of eukaryotic cells suggests that loss of clonogenic capacity and cell death I Italicized, three-letter code refers to the gene (e.g., ruf); three-letter code with first letter in uppercase refers to the protein (e.g., Raf).

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result from damage to the structure and function of genomic DNA (Painter, 1980). T h e clonogenic assay for studying responses of cells to radiation is based on the method described by Puck and Marcus (1956). T h e effect of increasing doses of radiation on the clonogenic capacity is described by a radiation dose-survival curve (Alper, 1979), which generally consists of an initial curved component in the low dose range (the shoulder) and of an exponential component (the terminal slope). The single-hit, multitarget (target model) and the linear quadratic model are most commonly used to analyze cellular radiation survival. A graphic representation of the single-hit, multitarget model and the linear quadratic model of radiation survival are shown in Figs. 1A and 1B. T h e target model is based on the parameters Do and 6,where Do is the inverse of the terminal slope of the survival curve and fi, reflects the extrapolation of this slope to the ordinate (Fertil et al., 1980, 1988; Steele et al., 1983). Another parameter, D, is the measure of the shoulder of the survival curve as the terminal slope line intersects the abscissa (Withers, 1987). T h e linear quadratic model has two major parameters: a, the linear component characterizing the radiation response at low doses; and p, the quadratic component predominating at higher doses. T h e higher the value of a,the more linear is the response of cells to low doses of radiation and the more sensitive are the cells to the cytotoxic effects of X-rays (Hall, 1988). In addition, using a model-free parameter, the mean inactivation dose (b,the area under the survival curve plotted on linear coordinates; Fig. 1 C) has been employed as a measure of the intrinsic radiosensitivity of human cell lines (Fertil et al., 1984). Finally, a distinction between the relative radioresistant and radiosensitive phenotypes can be made by comparison of the values of the survival fraction following exposure to 2 Gy (SF,), the dose most usually delivered per session of radiotherapy (Fertil and Malaise, 1985). Parameters that describe the exponential component are used to define the radiosensitivity of cells, whereas those parameters that describe the shoulder reflect the capacity to repair radiation lesions and to restore clonogenicity. Quantitative evaluations of the cellular capacity to repair radiation-induced sublethal damage, potentially lethal damage, DNA single-strand breaks and DNA double-strand breaks are routinely performed employing the previously described and/or modified procedures (Elkind and Sutton, 1959; Philips and Tolmach, 1966; Belli and Shelton, 1969; Little, 1969; Kemp et al., 1984; Wlodek and Hittleman, 1987; Iliakis and Seaner, 1988; Iliakis et al., 1991). In this review, the definition of relative radioresistance (or radiosensitivity) of different cell types, o r transfectants derived thereof, applies to the slope and/or the shoulder of the radiation survival curve. Changes

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FIG. 1. AnalysiLof a standard radiation survival curve using the multitarget model (A), the linear quadratic model (B), and the concept of mean inactivation dose (D)(C) (adapted from Fenil and Malaise, 1985).

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in these regions of the curve may result in greater (or lesser) survival after radiation exposure.

B. CLONAL NATUREOF THE RADIATION RESPONSE Clonal heterogeneity has been reported in both normal and neoplastic cells. This phenomenon includes such diverse features as tumor histology, metastatic phenotype, growth characteristics, antigens, and transplantability (Poste et al., 1981; Brattain et al., 1981; Rubin et al., 1983; Heppner, 1984). Heterogeneity of the radiobiological response(s) among clonally derived cell lines has been the focus of several investigations (Hill et al., 1979; Leith et al., 1982; Weichselbaum et al., 1988; Kasid et al., 1989~).These studies have reported significant variations in the radiation survival parameters of clonally derived cell lines representing such diverse cell types as colon and lung carcinomas, human squamous cell carcinomas, and NIH/3T3 cells. Earlier reports have revealed that the cotransfection of human tumor DNA and pSV2Neo plasmid DNA into NIH/3T3 cells results in G418resistant NIH/3T3 clones that demonstrate a spectrum of radiation responses ranging from a relatively radioresistant phenotype (Do = 2.28 Gy) to a relatively radiosensitive phenotype (Do = 1.36 Gy) compared to the untransfected parental NIH/3T3 strain (Do = 2.02 Gy) (Kasid et al., 1989~).However, heterogeneity in radiation response was also observed when the untransfected single cell-derived NIH/3T3 clones were studied (range, Do = 1.06 Gy to 2.38 Gy) (Kasid et al., 1989~).In a recent report, the y-radiation survival of Syrian hamster (SHOK) cells was shown not to be affected by transfection of the neomycin gene (Suzuki et al., 1992). The above studies are in contrast to an earlier report suggesting that transfection of a neomycin resistance marker and clonal selection could impart radioresistance to both normal and tumor cells (F’ardo et al., 1991). In the latter report, these investigators also did not find significant heterogeneity in the radiation response of clonally derived untransfected primary rat embryo cells or glioblastoma cells. Because clonal cell lines derived from both NIH/3T3 and human tumor cell populations exhibit sufficient heterogeneity in radiation survival responses to make interpretation of oncogene effects exceedingly difficult (Leith et al., 1982; Weichselbaum et al., 1988; Kasid et al., 1989c), it seems reasonable to circumvent the heterogenous component of the radiation response phenotype by using pooled transfectant cell populations for studies of the effects of oncogenes on radiation survival response (Kasid et al., 1989a,b; Pirollo et al., 1989). However, in several reports discussed in this review, oncogene-transfected clonally derived

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cell lines have been used to investigate changes in radiation sensitivity as a function of oncogene expression. Therefore, for the sake of simplicity, this review will be based solely on the interpretations of the radiation survival data as they have been reported in the literature.

111. Human and Rodent Cell Model Systems The initial approach used to identify the genetic factors associated with relatively radiation-resistant cells both from a human squamous cell carcinoma and from nontumorigenic skin fibroblasts (NSFs) of certain cancer-prone individuals was based on cotransfection of the representative human DNA and pSV2Neo plasmid DNA into NIH/3T3 cells followed by G418 selection and screening of the G418-resistant NIH/3T3 transfectant clones for the presence of human counterparts of the known oncogene sequences (Kasid et al., 1987a; Chang et al., 1987; Pir0110 et al., 1989). Subsequently, cloned cDNA of the candidate human protooncogene (raf-1) was expressed in human squamous carcinoma cells in an attempt to determine the phenotypic changes in the recipient human cells (Kasid et al., 1989a). A similar approach was used in studies using other human cell lines and rodent cell lines in order to determine the radiobiological consequences of the expression of a variety of protooncogenes (Pirollo et al., 1989, Table I). This section will (a)summarize the various cell systems that have been studied to date and (6) identify the candidate oncogenes that appear to have a potential role in the regulation of radiation resistance. A. RADIORESISTANT HUMAN SQUAMOUS CELLCARCINOMAS Relatively radioresistant tumor cell lines (SQ-2OB, SCC-35, JSQ-3) were established in culture from squamous cell carcinomas of head and neck origin following the full course of radiotherapy (Weichselbaurn et al., 1989). DNA-mediated gene transfer was used to investigate the genetic factors associated with these tumor cells. The human raf-1 sequences were found in the NIH/3T3 clones transfected with these DNAs. A majority of the NIH/3T3 transfectants were highly tumorigenic in athymic mice (Kasid et al., 1987a, 1993). Significantly, the NIH/3T3 transfectant clone lacking the kinase region (cl 21) was nontumorigenic, as were the control untransfected NIH/3T3 cells (Kasid et al., 1987a). The identification of the loss of the regulatory domain and retention of the kinase domain in the highly tumorigenic clones supports the hypothesis that deletion of the regulatory region results in the

20 1

ONCOGENIC BASIS OF RADIATION RESISTANCE

TABLE 1

ONCOGENE EFFECTS ON RADIATION RESPONSE Oncogeneu v-abl

Cell type used for oncogene transfection

N 1H/3T3

Changes in responseb NC

Sklar el al. (1986); Pirollo et al. (1989) FitzCerald et al. (1991)

t

Suzuki et al. (1992)

NlH/3T3 and murine hematopoietic cells, 32D c13 c-cot

Syrian hamster OsakaKanazawa (SHOK) cells

FitzGerald et al. ( 1990) Suzuki et al. (1992) Pirollo et al. ( I 989)

32D c13 SHOK cells v-fes V Y P

c-fm

v-fos v-mas c-myc

References

NIH/3T3 SHOK cells

Suzuki et al. (1992)

N I H13T3 32D c13

Sklar et al. (1986) FitzGerald et al. (1991)

NI H/3T3

Sklar (1988) FitzGerald et al. (1991)

N IH/3T3 NlH/3T3 SHOK cells N 1H /3T3 Rat embryo cells (REC)

FitzGerald et al. (1990) Pirollo et al. (1989) Suzuki et al. (1992) Pirollo et al. (1989) Ling and Endlich (1989) McKenna et al. (1990a) Kasid el al. (1989b)

lmmortalized human bronchial epithelial cells (Beas-2B) SHOK cells

Suzuki et al. (1992)

v-myc

32D c13

FitzGerald et al. (1991)

c-raf- 1

Human SCC, (SQ-2OB) NIH/3T3 Beas-PB Beas-2B

Kasid et al. (1989a) Pirollo et al. ( I 989) Kasid el al. (1989b)

c-raf-I and c-myc c-raf-I (AS) c-H-rm

SQ-20B NlH/3T3 Transformed human embryo retinal cells (HER)

Kasid et al. (1989b)

.1

Kasid et al. (1989a)

NC

Sklar (1988) Pirollo at al. (1 989); Samid et al. (1991) Grant et al. (1990)

t

NC

(continued)

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TABLE I (Continued ) Oncogenea EJ-Tu

v-H-T~

Cell type used for oncogene transfection

Changes in responseb

N I H13T3

t

REC

f (m)

Human mammary epithelial cells, (HBL 100) Immortalized human keratinocytes (HaCaT)

NC

NIH/3T3

References Sklar (1988); Pirollo et al. ( 1989) Ling and Endlich (1989); McKenna et al. (1990a) Alapetite el al. (1991)

NC

Mendonca et al. (1991)

t t (Id)

Sklar (1988) FitzGerald et al. (1990) Suzuki et al. (1992)

SHOK cells

NC

NIH/3T3 Rat kidney epithelial cells SHOK cells

J.

NC

N I H13T3

t

HER SHOK cells REC

NC

REC

t

McKenna et al. (1990a)

V-Sic

32D c13

NC

FitzGerald et al. (1990)

v-STC

N I H13T3 32D c13 Rat fibroblast cells (LA-24) Multidrug-resistant LA-24 cells

J.

t (Id)

NC

FitzGerald et al. ( 1990) FitzGerald et al. (1990) Shimm el al. (1992)

t

Shimm et al. (1992)

v-K-T~s

N-rm

EJ-rm and c-my EJ-rm and v-my

t

t t

Sklar (1988) Harris et al. (1990) Szuki et al. (1992) FitzGerald et al. (1985); Sklar (1988) Grant et al. (1990) Suzuki et al. (1992) Ling and Endlich (1989)

Oncogenes are alphabetically arranged on the basis of their three-letter code. AS, antisense cDNA. t , evidence suggesting increase in relative radiation resistance (Do or D, value); 1, evidence suggesting decrease in relative radiation resistance (Do value); NC, no change reported compared to the experimental control; f (m). moderate increase in relative radioresistance; t (Id). increase in radioresistance reported at low dose range y-irradiation.

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catalytic activation of the kinase domain (Kasid et al., 1987a; Pfeifer et al., 1989a; Stanton et al., 1989; Heidecker et al., 1990). Although the identification of human m f - 1 sequences in the NIH/3T3 transfection assay has been reported using a variety of tumor DNA samples (Shimizu et al., 1985; Fukui et al., 1985; Ishikawa et al., 1986; Stanton and Cooper, 1987), the presence of human ruf-1 sequences in all of these human a h containing NIH/3T3 transfectant clones derived from transfection of these radioresistant tumor cell-derived DNAs is remarkable. Because the clonally derived NIH/3T3 cell lines (untransfected) present a heterogenous population in terms of their radiation sensitivities (Do values), a direct correlation between activated raf-1 and radiation response was not feasible in the above-described NIH/3T3 transfectants (Kasid et al., 1989~). Given the complex nature of cellular radiation sensitivity, one way to demonstrate a correlation between raf- 1 activation and resistance to ionizing radiation in tumor cells is to perform radiation survival analysis on a pooled cell population in which rafi 1 expression has been inhibited by transfection of antisense human rafil cDNA. Indeed, this antisense RNA approach has been successfully used to demonstrate that the down-regulation of endogenous rafi 1 expression leads to decreased tumorigenicity and enhanced radiation sensitivity of human squamous carcinoma-derived cells, SQ-20B (Kasid et al., 1989a) (Fig. 2). These data provide evidence for the role of raf-1 function in radiation resistance. Furthermore, these studies demonstrate that the antisense vector-based strategy to inhibit the biological outcome of a specific gene function is also applicable to the radiation response phenotype. T h e modulatory effect on radioresistance due to antisense c-raf- 1 expression was evident only during the early passages in culture, as has been observed by other investigators using antisense RNA constructs (Bolen et al., 1987). Nevertheless, modifications of the antisense RNA strategy using inducible vectors or inhibition of Raf- 1 function by antisense deoxyoligonucleotides (Kasid et al., 1991a) are promising approaches for further investigations into the oncogenic basis of radiation resistance. B. SKINFIBROBLASTS FROM

A

CANCER-PRONE FAMILY

T h e cancer family syndrome originally described by Li and Fraumeni (1969)is characterized by a constellation of tumor types including breast carcinoma, soft tissue sarcoma, brain tumors, osteosarcoma, and leukemias. These diverse neoplasms, which occur in a dominantly inherited

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0.005

0.001

I -

0 100

300

500

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900

Radiation Dose (cGy)

FIG. 2 . Clonogenic radiation survival curves for c-raf-1 (S) and c-raf-1 (AS) cDNAtransfected SQ-2OB cell populations (adapted from Kasid el al., 1989a). The experimental points are plotted &SEM. The Do values were 310 and 191 cCy for c-raf-I (S) cDNA and for c-raf-1 (AS) cDNA-transfected SQ-POB cell populations, respectively.

pattern, develop at an early age with multiple primaries appearing in the same individual. In some instances they appear to be related to carcinogenic exposures, including ionizing radiation. Recently, inherited germline mutations in the tumor suppressor gene p53 were simultaneously identified by two different groups in a total of six different LiFraumeni families (Malkin et d.,1990; Srivastava et al., 1990). These mutations, which are located in a highly conserved region of the gene, are believed to represent the primary inherited defect predisposing these individuals to develop cancer. One particular Li-Fraumeni family has been studied more exten-

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sively than others. This specific family, originally described by Blattner et al. (1979), involves 18 affected descendants of a single individual through six generations. Neoplasms in 3 members of the family may have been induced by occupational exposure or therapeutic radiation. One member developed polycythemia Vera after working for 5 years in a factory producing heavy water, a second family member with lung adenocarcinoma worked in a foundry, and an osteosarcoma was diagnosed in a third individual within the field of radiotherapy for an earlier neurilemmoma. In addition to the inherited germline mutation in codon 245 of p53 identified in this family (Srivastava et al., 1990), examination of nontumorigenic skin fibroblast (NSF) cell lines from individuals in the cancer-prone lineage revealed a three- to eightfold elevation in the level of c-myc expression relative to that found in unrelated control fibroblast cells (Chang et al., 1987). Moreover, by means of the NIH/3T3 transfection assay, the presence of an activated raf-1 oncogene has also been detected in the NSFs from at least one family member (Changet al., 1987). It is also noteworthy that the NSF cell lines from most members of this family have been found to display the unusual property of resistance to the killing effects of ionizing radiation (Bech-Hansen et al., 1981) (Fig. 3). Five of the family members examined demonstrated an increased level of radiation resistance relative to the normal controls. In one instance multiple cell lines representing biopsies taken from the same individual but at different times were included in the study. T h e differences in the D,,values were statistically significant (P< 0.001) with the fibroblast line from individual IV-19 having one of the highest levels. Furthermore, no correlation was found between the presence of the inherited p53 mutation and the radiation-resistance phenotype observed in individuals in this family. These results support the notion that the radiation resistance may be one of the inherited defects in this specific cancer-prone family, although this clearly is not the case for all LiFraumeni families (Little et d., 1987). Moreover, heterogeneity does exist among these kindred inasmuch as not all of the families previously identified as having Li-Fraumeni syndrome were found to contain a germline mutation in the tumor suppressor gene p53 (Santibanez-Koref et al., 1991). An association between the radiation-resistant phenotype present in these family members and the activated r a . 1 gene was demonstrated when a tertiary NIH/3T3 transformant derived from DNA of the NSF cell line with one of the highest levels of radiation resistance was assessed for its resistance to killing by y-radiation. This human raf- 1-containing mouse cell line demonstrated an increased level of radiation resistance

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I

I

I

I

I

IIL I

P

PV

Br

I

BT BT

BT

Br

ss

# :Single primary cancer

9I: BT

0s

in deceased female : Double primary cancer in proband

FIG.3. Partial pedigree of a specific cancer-prone family with Li-Fraumeni syndrome (modified from Bech-Hansen et al., 1981). Shown here is a branch of a much larger pedigree that traces cancers over five generations in three separate lineages from a woman who died of breast cancer in 1865 (Blattner el al., 1979). Individuals whose normal skin fibroblast cell lines were found to demonstrate an increased level of radioresistance are designated “R.”N, normal level of radiation response; OS, osteogenic sarcoma; S S , soft tissue sarcoma; BT, brain tumor; Br, breast cancer; PV,polycythemia Vera; Le, leukemia; Co, colon cancer; NL, neurilemrnoma.

relative to that of the untransfected NIH/3T3 cells, a level which approximated that observed in the radiation resistant parental NSF cell line (Pirollo et al., 1989)(Fig. 4).This report of the relationship between activated rafand radiation resistance is among the first to link this phenotype to the function of a specific oncogene. Two additional features of the NSF cell lines from members of this cancer-prone family that may be related to its radiation-resistant phenotype have been examined. The first is the level of activity of the topoisomerases in these cells. The activities of both topoisomerase I and I1 in the NSFs of the family were examined (Cunningham et al., 1991). The activity of topoisomerase I1 was found to be elevated in the radiation-resistant cell lines of this family but not in that of the non-radiationresistant cell line derived from a normal spouse (V-7).Topoisomerase I activities were comparable in all of the lines tested. The second feature is the presence of an altered DNA de novo synthesis in the NSFs of family members. The radioresistant NSF cell lines from the proband (VI-2)and his great uncle (IV-19),which both sustain and repair radiogenic DNA damage at rates comparable to those of non-

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600-.

cA

ul

550 --

U

500-

3

% 450-

0'

400-

350-

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T 111

0 T

OT

t

T V 1

rn

0-NSF 2800 A-3T3lraf 0 3T3lmos Q A-3T3IEJ-ras 0-3T3lLTR- ras

-

0- NIH3T3 A 3T3lmyc V-3T3Ifes 3T3labl

-

-

FIG.4. Scattergram of the D I Ovalues of various NlH/JTS-transformed cell lines demonstrating an increased level of radiation resistance for cell lines transformed by the raf, mos. or ras oncogene, relative to the parental NIH/3T3 cell line (modified from Pirollo et al., 1989). Each point represents the mean of two to five experiments 2 standard error. T h e error bars for NIH/3T3,3T3/abl, 3T3/mos, and 3T3IEJ-ras are absent due to the fact that the standard errors for these four cell lines (k3.6, 26, 2 7 , and 2 5 , respectively) are too small to be visualized within the parameters of the graph. NSF 2800 is the radioresistant normal skin fibroblast cell line derived from individual IV-19 in the family pedigree (Fig. 3) and is the cell line from which NIH13T3 transformant 3T3lrafwas derived (Chang et al., 1987).

radiation-resistant fibroblast cell lines, possess what has been described as an error-prone semiconservative DNA synthesis mechanism (Paterson et al., 1985). After treatment with y-rays (60Co), these cells displayed a longer lag time prior to initiation of synthesis and sustained a higher level of synthesis for a protracted period of time when compared to nonradiation-resistant fibroblast cells. It is of interest to note that the abnormality in DNA synthesis evident in these radiation-resistant fibroblast cell lines is the exact opposite of that seen in radiation-sensitive AT cells where DNA synthesis is appreciably reduced relative to normal cells (Houldsworth and Lavin, 1980). Therefore, this Li-Fraumeni family is a naturally occurring system that implicates the mf oncogene in the genesis of radioresistance. Since the radiation-resistant NSF cell lines are not tumorigenic, this model also provides evidence that radiation resistance and transformation are not necessarily coincidental phenomena (see also Section V). Furthermore, it suggests that events in the nucleus, such as those involved in DNA conformation, synthesis, and repair, may be important factors in cellular radiation resistance.

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C. NIH/3T3 TRANSFECTANTS The earliest report of a possible link between an activated oncogene and a radiation-resistant phenotype in NIH/3T3 cells showed that transfection of the human N-ras oncogene was able to increase the level of relative resistance of the recipient cells at a relatively high radiation dose rate (200 cGy/min) (FitzGerald et al., 1985). Subsequent studies demonstrated that the effect of activated oncogenes on the radiation-resistance level of NIH/3T3 cells is not a generalized phenomenon but is particular to specific oncogenes (Sklar et al., 1986; Sklar, 1988; Pirollo et al., 1989; Suzuki et al., 1992); this will be discussed in greater detail below. The presence of v-H-ras, v-K-ras, EJ-ras, and N-ras oncogenes were found to significantly increase the radioresistance levels of NIH/3T3 cells, whereas NIH/3T3 cells transformed by either vfm or v-a61 did not exhibit changes in radiation sensitivity relative to the untransfected cells (Sklar et al., 1986; Sklar, 1988). A similar effect on the radiation response of NIH/3T3 cells was also demonstrated by overexpression of the normal H-ras protooncogene (Pirollo et al., 1989). Moreover, a significant degree of radiation resistance was conferred on NIH/3T3 cells expressing members of the protein-serinelthreonine kinase family (rufand mos) (Fig. 4). The effects of oncogenes appear to vary depending on the dose rate of y-radiation employed. Although the experiments using a high dose rate of the y-radiation (60Co; = 120 cGy/min) demonstrated no change in the radiation response of NI H/3T3 cells transfected with v-abl, vfm, o r v-fos (Pirollo et al., 1989), other reports using low-dose-rate y-radiation ( 13’Cs; 5 cGy/min) have indicated a significant increase in the radiation resistance of NIH/3T3 cells expressing these oncogenes (FitzGerald et al., 1990, 199 1). T h e precise mechanism(s) and significance underlying these differential effects on radiation response due to the differences in the dose rate of y-radiation employed are unclear at present. D. OTHER CELLLINES

T h e synergistic effect of EJ-ras and c-myclv-myc on the radiation-resistant phenotype has been reported in studies using primary rat embryo cells, REC (Ling and Endlich, 1989; McKenna et al., 1990b). By itself, EJ-ras had only a limited effect on the radiation response of RECs, whereas the myc gene had no effect (McKenna et al., 1990a,b). Together these two oncogenes exhibited a significant increase in radioresistance over the parental cells. These observations suggest the possibility of a putative interaction among the products of the ras and m y oncogenes in signaling mechanisms underlying the radioresistant phenotype.

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I n a recent report, resistance to y-rays was conferred by the introduction of v-mos, c-cot (both genes encoding cytoplasmic protein serine kinases), o r N-ras gene into Syrian hamster (SHOK) cells (Suzuki et al., 1992). Interestingly, the radiation sensitivity of these cells did not change upon the transfection of the v-fgr, c-myc, v-erb-B, Ha-ras, or K-ras gene. T h e morphological transformation associated with the induction of v-src did not correlate with the radioresponsiveness of rat fibroblast cells (LA-24). However, in the multidrug-resistant clones of these rodent cells, a significant increase in radioresistance has been reported to correlate with the induction of v-src (Shimm et al., 1992). Murine hematopoietic progenitor cells, 32D c13, have also been analyzed for the oncogenic effects on radiation response using low-dose-rate y-radiation. These experiments suggest that with the exception of v-szi, the other oncogenes tested, namely v-erb-B, v-abl, v-src, c$m, and v-myc, are all able to induce radiation resistance in 32D c13 transfectant cells (FitzGerald el al., 1990, 1991). T h e increase in radiation resistance of immortalized human bronchial epithelial cells (Beas-2B) by the expression of raf-1 has also been demonstrated (Kasid et al., 1989b). These reports suggest that the increased expression of raf- 1 is sufficient to increase radioresistance in the nontumorigenic Beas-2B cells, whereas c-myc expression does not change the radiation dose response. In addition, Beas-2B cells transfected with a combination of raf-1 and c-myc genes demonstrated no further increase in the level of radiation resistance (Do value). However, this does not rule out possible synergism between rafand myc in other cell systems. Taken together, the studies to date suggest that Raf-1 plays a dominant role in the radiation-resistant response of both human and rodent cells. Finally, the role of the ras oncogene in the radiation response of human cells is somewhat intriguing. To date, three different human cell model systems have been examined in an effort to elucidate the role of the ras gene product in the radiation response of human cells. T h e N-ras and c-H-ras genes did not seem to alter the radiation sensitivity of transformed human embryo retinal cells (Grant et al., 1990) and EJ-ras transfection and expression had no effect on the radiation response of human mammary epithelial cells (HBL 100) and immortalized human keratinocytes (Alapetite et al., 1991; Mendonca et al., 1991). T h e complex basis of the ras gene-induced effects on radioresistance is also evident from another study in which the radiosensitization of rat kidney epithelial cells containing K-ras has been reported (Harris et al., 1990). Among other possibilities, the differentiation state of the cells and/or a cooperation between another gene and ras may be required to modulate

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the radiosensitivity of these cell types. Alternatively, a high level of inherent radioresistance may be a limiting factor in certain cell types. Moreover, such reports serve to remind us of the fact that these types of studies can be complicated by both the methodology and the biology of the system.

IV. Mitogenic Signals and Radiation Response The normal counterparts of many of the known oncogenic products have been shown to interact with one another as components of a proposed signal transduction pathway that serves to transmit messages from the cell membrane to the nucleus (Weinstein, 1988a). On the basis of antibody-blocking experiments, Raf- 1 has been placed downstream of Ras in this pathway (Smith et al., 1986; Rapp et al., 1988b; Morrison et al., 1989).As discussed earlier, the r a . 1 oncogene has been implicated in the expression of the radiation-resistant phenotype (Chang et al., 1987; Kasid et al., 1987a, 1989a,b; Pirollo et al., 1989). Additionally, since oncogene products upstream of Ras and Raf in the proposed signaling pathway are able to induce the resistant phenotype and reports from several other laboratories have indicated that activated ras oncogenes, or a combination of ras and myc oncogenes, can influence the radiation resistance level of cells into which they are transferred (Table I), it appears that the expression of radiation resistance may be under the control of a similar type of signal transduction mechanism. Protooncogenes have been shown to code for growth factors and growth factor receptors such as platelet-derived growth factor (PDGF) B-chain, truncated epidermal growth factor (EGF) receptor, fibroblast growth factor (FGF)-related growth factor, colony-stimulating factor (CSF-1) receptor, and nerve growth factor (NGF) receptor (Hunter, 1991).The effect of the oncogenefms, which codes for the mutant CSF-1 receptor protein-tyrosine kinase on radiation response has been examined. This gene has been found to be incapable of modulating the radiation sensitivity of NIH/3T3 cells (Sklar et al., 1986; Sklar, 1988) except at low doses of radiation (FitzGerald et al., 1990). Members of the family of nonreceptor protein-tyrosine kinases seem to be incapable of effecting the level of radiation resistance. The oncogenesfes, abl, and fgr did not alter the radiosensitivity of the recipient NIH/3T3 or Syrian hamster (SHOK) cells (Pirollo et al., 1989; Suzuki et ad., 1992).The exception appears to be the Src protein kinase. The v-src oncogene confers radioresistance on murine hematopoietic cells 32D c13 (at a low-dose-rate y-radiation) (FitzGerald et al., 1990). However, Src protein-tyrosine kinase induction did not increase the radioresistance in

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rat fibroblast cells (LA-24) and appeared to increase the sensitivity of NIH/3T3 cells (FitzGerald et al., 1990). Interestingly, a significant radioresistance was noted in the multidrug-resistant variants of LA-24 cells upon induction of the Src kinase (Shimm et al., 1992). G proteins, which are involved in the reversible exchange of GTP for GDP with concomitant activation of an effector protein, also have a role in the signal transduction pathway. T h e ras p2 1 protein functions as a G protein (Trahey and McCormick, 1987; Vogel et al., 1988) and has been implicated in the activation of phospholipase C (Berridge and Irvine, 1984; Chabre, 1987; Cockroft, 1987; Marshall, 1987; Katain and Parker, 1988). T h e role of the ras gene in radiation resistance has been studied by several investigators, using not only the NIH/3T3 transfection assay (FitzGerald et al., 1985, 1990; Sklar, 1988; Pirollo et al., 1989; Samid et al., 1991), but also the primary rat embryo fibroblast cells (REC) (Ling and Endlich, 1989; McKenna et al., 1990a), Syrian hamster cells (Suzuki et al., 1992), rat kidney epithelial cells (Harris et al., 1990), and human cells (Grant et al., 1990; Alapetite et al., 1991; Mendonca et al., 1991). T h e presence of any member of the ras family, activated through either mutation o r overexpression, was sufficient to significantly increase the level of the radiation resistance in the recipient NIH/3T3 cells. N-rtls expression but not Ha-ras or Ki-ras expression was able to increase the radioresistance in Syrian hamster (SHOK) cells Suzuki et al., 1992), and only a moderate increase in radioresistance of REC was noted following the transfection of EJ-ras (Ling and Endlich, 1989; McKenna et al., 1990a,b). However, the cotransfection of EJ-ras and c-myclv-myc led to a significant increase in the relative radioresistance of REC (Ling and Endlich, 1989; McKenna et al., 1990b). In contrast, rat kidney epithelial cells containing K-ras had increased radiation sensitivity with ras activation. Human cells, however, did not show changes in the radiosensitivity in response to the ras expression. Although a cooperation between oncogenes may be required for the development of in vitro radioresistance in human cells, the evidence is not yet available. T h e cytoplasmic protein-serine/threonine kinases play a central role in signal transduction and are also implicated as one focal point in the pathway to the radiation-resistant phenotype. The prototype for such serine/threonine kinases, protein kinase C (PKC), is a principal effector for signaling mediated by phosphotidylinositol-4,5-biphosphatehydrolysis and is the vehicle by which a number of tumor promoters act (Weinstein, 1988b). Based on Raf-1 inhibition experiments, it is suggested that PKC-dependent mitogenic signals are transduced by PKCmediated activation of Raf-1 kinase in NIH/3T3 cells (Kolch et al., 1991). However, the PDGF effects on Raf-1 are seen in fibroblasts that have

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been chronically treated with a tumor promoter to down-regulate PKC (Morrison et al., 1988). Therefore, it appears that PKC is not always an effector for the activation of Raf- 1 kinase. However, the possibility exists that PKC may also be affecting the cellular response to y-radiation (Weichselbaum et al., 1991). Most important, cot, mos, and the raf family of oncogenes all encode protein-serine kinases (Aoki et al., 1991; Seth and Vande Woude, 1988; Rapp et al., 1988b) and all have been associated with the acquisition of radiation-resistant phenotype (Kasid et al., 1989a,b; Pirollo et al., 1989; Suzuki et al., 1992). Raf-1 protein kinase appears to be at a central location in the signaling pathway to radiation resistance. An association between the raf- 1 gene and the radiation-resistant phenotype in the NSFs from a specific cancer-prone family with Li-Fraumeni syndrome has been established (Pirollo et al., 1989). Transfection of antisense human raf-1 cDNA into radioresistant human squamous carcinoma cells leads to the down-regulation of endogenous raf 1 expression, delayed tumor growth, and enhanced radiation sensitivity (Kasid et al., 1989a). In addition, the transfection of human raf- 1 cDNA into immortalized human bronchial epithelial cells (Beas-2B) is sufficient to increase the radioresistance of Beas-PB-raf transfectants (Kasid et al., 1989b). These studies suggest a close link between the radiation-resistant phenotype and the function of raf-1 in human cells. Increased phosphorylation and elevated enzymatic activity of Raf- 1 protein kinase have been demonstrated in numerous cell types tested in response to a variety of ligands (Rapp, 1991). In fact, Raf-1 has been found to be complexed with at least two growth factor receptors, EGF-R (App et al., 1991) and PDGF-R (Morrison et al., 1989). Oncogenes shown to have homology to one of the EGF receptors and to PDGF (HER-2 and sis, respectively) have been shown to increase radiation resistance of NIH/3T3 cells (Pirollo et al., 1991). Further evidence for the importance of Raf-1 protein kinase in the signaling pathway leading to the radiation resistance is suggested by the report on the radioprotective effects of granulocyte macrophage-colony-stimulating factor (GM-CSF) (Waddick et al., 1991), which has been shown to increase the activity of Raf-1 protein kinase (Carroll et d., 1991). Therefore, it appears that the functional activation of Raf-1 protein kinase either via direct alteration in the product itself or via modulation of the upstream signals may be sufficient or even necessary for the induction of the molecular/biochemical events leading to radioresistance. The ultimate targets of signal transduction in the nucleus are probably the least understood part of the pathway. However, there are some

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indications of what may be occurring there. The mitogenic signal(s) received by Raf-1 protein kinase may be forwarded to the nucleus by phosphorylation of transcription factors such as AP-1, which is an association of the products of protooncogenes fos andjun (Chiu et al., 1988; Rauscher et al., 1988; Sassone-Corsi et al., 1988; Bruder et al., 1992). Inactivation of Raf- 1 protein kinase reportedly blocks the transcription of a reporter gene from the promoter containing the AP- 1 DNA binding motif (Bruder and Rapp, 1991; Bruder et al., 1992). These studies indicate that AP-1 requires Raf-1 protein kinase for the transactivation of transcription. T h e effect ofjun on intrinsic radioresistance is not known but fos has no effect on the radiation response of NIH/3T3 cells at low doses (FitzCerald et al., 1990). Experiments using purified components suggest that the Jun but not the Fos component of AP-1 is phosphorylated by Raf-1 (Heidecker et al., 1992). The nuclear oncogene, myc, coding for Myc protein which is downstream of Raf- 1 in the proposed mitogenic signal transduction pathway, is unable by itself to increase the level of radiation resistance in NIH/3T3 cells (Pirollo et al., 1989). Similar results were obtained using primary rat embryo cells (REC) and human bronchial epithelial cells (Beas-2B) (McKenna et al., 1990a; Kasid et al., 1989b). However, when both ras and myc genes were introduced into REC, a synergistic increase in radiation resistance was noted (Ling and Endlich, 1989; McKenna et al., 1990a). In transfection experiments utilizing Beas-2B cells, cotransfection of the raf and myc genes apparently did not induce a synergistic level of radiation resistance. However, this does not necessarily rule out the possibility of cooperation between these two oncogenes. It is conceivable that there may be an upper limitation to the level of detectable in vitro radioresistance (Do value), such that, in this instance, any contribution by Myc may not have been discernible. The support for the combined role of c-myc and c-raf-1 in radioresistance is derived from several lines of investigations. Amplification of c-myc has been associated with an in vitro radiation resistance of a variant of small cell lung carcinoma (oat cell) (Carney et al., 1983; Little et al., 1983), as well as with certain human lung cancer cell lines (Carmichael et al., 1989). Interestingly, a high level of expression of the raf-1 gene, along with a concomitant activation of Raf-1 protein kinase activity, has been found in approximately 60% of all lung cancers (Rapp et al., 1988a). Moreover, an activated raf-1 oncogene was identified via the NIH/3T3 transfection assay, as the transforming gene in a human lung carcinoid (CA1-154) (Stanton and Cooper, 1987). In this same vein, the radiation-resistant NSF cell lines from a specific cancer-prone family with Li-Fraumeni syndrome exhibit both an elevated level of c-myc

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expression and the presence of an activated raf-1gene, as suggested by the NIH/3T3 transfection procedure. Furthermore, c-my expression is known to be regulated at the level of the cell cycle. Therefore, these data indicate two possibilities: (a)a cooperativity toward radioresistance may exist between the raf and the m y genes and (6) genes such as m y whose expression is also regulated in a cell cycle-dependent manner may modulate in vitro radiosensitivity. The reports available so far suggest the possibility of a signal transduction pathway for radiation resistance analogous to that suggested for cell growth and proliferation or differentiation. Although many of the intermediate steps in this pathway have not yet been identified, there is strong evidence that the cytoplasmic serinelthreonine kinases, particularly Raf-1, play a central role. Several possibilities arise by which multiple signals may interact to influence the radiation-resistant phenotype. In one instance, proteins that already exist in the activated form interact with one another to generate a cell population that is selected for after exposure to radiation. Alternatively, exposure of cells to radiation precipitates events in the nucleus through changes in chromatin. These changes may trigger a cascade of events similar to those described above, leading to cell survival. I n addition, the ability of cells to delay progression through the cell cycle following DNA damage (G, and/or G, arrest) may be an important determinant of cell survival. It is also possible that the key component of the signal directly involved in radioresistance is initiated after exposure to y-rays. For example, radiation is known to induce programmed cell death (apoptosis) (Kerr and Searle, 1980).More recently, the extent of radiation-induced apoptosis has been shown to differ markedly between radiosensitive and radioresistant tumors and correlated with their respective response to local tumor irradiation (Stephens et al., 1991).It seems very likely that the products of certain oncogenes may counteract radiation-induced apoptosis. It is tempting to speculate that this is accomplished by an oncogene-activated effector protein(s), which may direct the normal replication machinery to read through the damage, thereby allowing the cells to proliferate. In support of the role of oncogenes in apoptosis, evidence is beginning to emerge that suggests a role for v-Raf in the suppression of this physiological control mechanism (Troppmair et al.,

1992). Since much is still unknown about the oncogenic interaction(s) in signal transduction, and about the biochemical and physiological bases of radioresistance, the working hypothesis put forth here may likely be modified with the advancement of our knowledge.

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A majority of the relevant data in the literature is consistent with the above hypothesis. However, some discrepancies do exist. The c-myc oncogene, which was shown not to increase the radiation resistance level of NIH/3T3 cells, rat embryo cells (REC) and human bronchial epithelial cells (Pirollo et al., 1989; Kasid et al., 198913; McKenna et ad., 1990a) did so in one study using RECs (Ling and Endlich, 1989) and also in one study using the hematopoietic progenitor cell line, 32D c13 (albeit at low dose rate only) (FitzGerald et al., 1991). NIH/3T3 cells transformed by abl or fms were found not to be radiation resistant (Sklar et al., 1986; Sklar, 1988; Pirollo et al., 1989). However, in the 32D c13 cells both oncogenes were able to induce radiation resistance, again at low dose rate of y-radiation (FitzGerald et al., 1991). Evidence from clinical studies indicates that different cell types have differing responses to radiation. In fact, studies, primarily with mice, demonstrated that in whole-body irradiation, the cells of the hematopoietic and the gastrointestinal systems are the most sensitive to the killing effects of ionizing radiation (Bond et al., 1965; Bond, 1969; Broerse and MacVittie, 1984). T h e observed effect of oncogenes on radiation resistance may also vary depending on the cell types used in the study (human vs mouse cells; fibroblasts vs epithelial cells or keratinocytes), further complicating the issue. Moreover, some cell types that display a significant level of relative radiation resistance (as measured by the relatively high Do value) may be inappropriate for use in such studies. In support of the latter argument, no change in the Do value of primary human keratinocytes (Do = 2.24 Gy) was observed following immortalization by SV-40/AD-12 virus (Do = 2.43 Gy) or subsequent transformation of the immortalized human keratinocytes by KiMSV infection (Do = 2.51 Gy) (Kasid et d., 1987b). T h e presence of discrepancies, such as those discussed above, underscore the complexity of the factors contributing to radiation resistance and the importance of future studies to achieve a better understanding of the molecular mechanisms involved.

V. Transformation and Radiation Resistance Based upon the above data, it is evident that the signal transduction pathways for mitogenesis and radiation resistance may have some elements in common. Although these signals may intersect with one another at some points, they obviously represent independent pathways that are neither identical nor even concurrent. Support for this is derived from the following reports. It has been well established that ras alone is incapable of transforming primary cells (REC); a cooperative

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oncogene which can immortalize the cells such as myc o r E1A is required for transformation. However, nontumorigenic ras-containing RECs were found to be more radiation resistant than the recipient RECs (Ling and Endlich, 1989). Further evidence of the separation of the two phenotypes is seen in a study of ras-transformed and phenotypically revertant NIH/3T3 cells. In these studies both the tumorigenic and the revertant, nontumorigenic NIH/3T3 cells expressed high levels of ras and exhibited an elevated level of resistance to radiation indicating that, although ras is clearly responsible for the radiation-resistant phenotype of these cells, perhaps another gene is necessary for the transformed phenotype (Contente et al., 1990; Samid et al., 1991). In addition, a distinction between transformation and radioresistance is provided by a report on rat fibroblast cells infected with a temperature-sensitive mutant of v-src (LA-24). In these studies, induction of v-src resulted in the morphological transformation of LA-24 cells but did not change their radiosensitivity, whereas in multidrug-resistant variants, the v-src induction caused a significant increase in radioresistance (Shimm et al., 1992). In the case of the ruf oncogene also, a division between oncogenic transformation and radiation resistance is evident. As mentioned earlier, the radiation-resistant NSF cell lines from a cancer-prone family with Li-Fraumeni syndrome are clearly nontumorigenic. Moreover, immortalized, nontumorigenic human bronchial epithelial cells transfected with the human r.f-1 cDNA are significantly more radioresistant compared to the untransfected cells or the cells transfected with the Zip-neo vector alone (Kasid et al., 1989b; Pfeifer et ul., 1989b). Therefore, it appears that the Raf-1 protein kinase has a dual role, one in mediating the malignant phenotype and a second in the DNA damage and repair cascade. The dissociation between the two pathways is also apparent from the data presented in Table I. Even though capable of a significant degree of transformation, the v-fm, v-fes, v-abl, o r v - f p oncogene does not seem to contribute to the radiation-resistant phenotype. T h e final destination for the signal in the nucleus may be the most critical determinant in differentiating between the two phenotypes. T h e challenge for future studies is to elucidate the mechanism(s) of this new role for certain protooncogene products, i.e., involvement in the cellular capacity to respond to damage and repair induced by y-radiation.

VI. Radiation-Resistant Phenotype: Cause or Effect Cellular radiation sensitivity is a complex function of diverse molecular, biochemical, genetic, and/or environmental factors (Russo et al.,

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1985; Thacker, 1986; Debenham et al., 1987; Cleaver, 1989). T h e functional involvement of oncogenic factors discussed above may represent only one important level of regulation of radiation response. It is also possible that certain oncogenic factors control the radiation response phenotype by regulating enzymes or substrates (effector proteins) involved directly o r indirectly in the DNA damage and repair system. Indeed, radioresistance/radiosensitivity of human tumor cells has been linked with a differential pattern of gene expression (Kasid et al., 1989d; Ramsamooj et al., 1992). A y-radiation-induced point mutation in c-K-ras has been shown to activate its oncogenic potential (Guerrero et al., 1984). It is not clear at this time whether radiation resistance is a consequence of radiationinduced structural changes in the oncogene(s). Recent reports indicate that ionizing radiation transiently induces certain genes at the transcriptional, translational, or post-translational level (Lambert and Borek, 1988; Boothman et al., 1989, 1991; Singh and Lavin, 1990; Sherman et al., 1990; Hallahan et al., 1991; Brach et al., 1991; Papathanasiou et al., 1991). These observations are highly significant in the context of this review, since some of the genes responsive to y-irradiation code for transcription factors (AP-1, Egr- 1) with a requirement for Raf-1 protein kinase (Bruder et al., 1992; Qureshi et al., 1991).This section reviews the data suggesting (a) that an association may exist between differential expression of the various gene products and radiation response, and (b) that ionizing radiation induces intracellular signaling events involving important growth-related biological molecules. A. MULTIFACTORIAL NATUREOF RADIATION RESPONSE: DIFFERENTIAL GENEEXPRESSION The possibility that multiple genetic factors are involved in the resistance o r sensitivity of human squamous carcinoma-derived cell lines was investigated using two-dimensional polyacrylamide gel electrophoresis (Kasid et al., 1989d; Ramsamooj et al., 1992). Based on the response to radiation therapy and in vitro radiation survival analysis, the tumor cell lines were classified as relatively radioresistant o r radiosensitive (Weichselbaum et al., 1989). A set of at least 14 different proteins was found to be preferentially expressed in each of the three radioresistant tumor cell lines (SQ-20B, SCC-35, JSQ-3; Do range, 2.3 to 2.5 Gy) compared to their expression in the radiosensitive tumor cells, and a set of at least 15 different proteins was specifically expressed in each of the three radiosensitive tumor cell lines (SQ-38, SCC-9, SQ-9G; Do range, 1.3 to 1.7 Gy), compared to their expression in the radioresistant tumor cells

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TABLE 11 IDENTIFICATIONOF MARKERPROTEINS IN RELATIVELY RADIORESISTANT A N D RADIOSENSIT~IVE HUMAN SQUAMOUS CARCINOMA-DERIVED CELLLINES^

Marker protein Radiation response phenotype

Molecular mass (kDa)

PI

Fold enhancement6

Radioresistant (RR) (Dorange, 2.3 to 2.5 Gy)

92

64 24 47 17

5.5 5.2 6.6 7.4 6.2

NA 17 16 10 10

Radiosensitive (RS) (Dorange, 1.3 to 1.7 Gy)

40 36 34 32 39

7.1 6.4 6.1 6.2 6.2

NA 45 10 9 6

Ramsamooj ct af. (1992). Fold enhancement value of the RR or RS protein represents quantitative difference observed in the signal compared to the value of corresponding protein spot in RS or RR cell type, respectively. NA, not applicable due to the undetectable levels in the other radiation response category. a

(Ramsamooj et al., 1992). A representative computer-assisted quantitative analysis of the 5 most significant marker proteins identified in each response category is shown in Table 11. Some of these proteins may represent candidates belonging to the effector-protein category. The role of these proteins in radiation resistance or sensitivity awaits their structural and functional characterization. Nevertheless, these findings suggest that the complexity of the radiation response phenotype may be due to the functional interaction of multiple proteins.

B. MOLECULARTARGETS OF IONIZING RADIATION A number of X-ray-inducible factors have been reported in recent years (Lambert and Borek, 1988; Boothman et al., 1989, 1991; Singh and Lavin, 1990; Sherman et al., 1990; Hallahan et al., 1991). Some of these genetic elements are immediately early genes (cjun,fos,junB, and egr-I) that are also induced rapidly in response to growth factors. The latter genes code for transcription factors AP-I and Egr-1. The induction of egr-1 and c-jun transcription by X-rays was attenuated upon inhibition of PKC by TPA treatment or by the protein kinase inhibitor H7 (Hallahan et al., 1991), suggesting that ionizing radiation induces a signal transduction pathway involving activation of PKC (Weichselbaum et al., 1991).

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Additional evidence in support of the y-radiation induction of PKCmediated signaling events is derived from studies based on the radiation-stimulated transcription of a reporter gene coding for chloramphenicol acetyl transferase (CAT) driven by the Moloney murine sarcoma virus long terminal repeat. Preincubation of X-ray-treated cells with TPA to down-regulate PKC abolished this activation process (Lin et al., 1990). Moreover, activation of transcription and the DNA binding activity of another transcription factor, NF-KB,was noted in response to y-irradiation of human myeloid leukemia cells (Brach et al., 1991). Since activation of NF-KB also occurs in response to UV-irradiation, another DNA-damaging agent, a reverse signaling pathway induced by DNAdamaging agents, that transduces signals from the nucleus to the cytoplasm has been proposed (Brach et al., 1991; Weichselbaum et al., 1991). T h e role for transcription factors in the regulation of transcriptional events is well known (Mitchell and Tjian, 1989). Given the functional significance of protein-serine/threonine kinases in radioresistance (discussed in the previous section), it may be of specific interest to note here that the Raf-1 protein kinase is required for the transcriptional transactivation function, via specific DNA binding sites of AP-1 and Egr-1 (Bruder et al., 1992; Qureshi et al., 1991). Cells that are deficient in Raf-1 protein kinase demonstrate impaired signaling in response to growth factors, deficiency in the induction of immediate-early genes (fos, junB and egr-1), and a block of transcription by the transcription factors AP-1, Ets-1, or Egr-1 (Rapp, 1991; Heidecker et al., 1992). Therefore, the obvious question is: What is the effect of the inhibition or activation of the Raf- 1 protein kinase in the y-radiation-induced transcription of genes coding for these transcription factors? The information gained from such studies may provide a significant advance in our understanding of the role of cytoplasmic kinases in the molecular and biochemical effects of y-irradiation, a biological consequence of which may be resistance to such toxic insults. The notion that ionizing radiation induces specific molecular signals also has support from studies of growth factors, cytokines, and cell cycle control genes. Radiation treatment releases growth factors similar to PDGF-a and FGF from vascular endothelial cells (Witte et al., 1989). Basic FGF (bFGF) has been shown to induce the repair of radiationinduced potentially lethal damage (Haimovitz-Friedman et al., 1991). Precoating of culture dishes with bFGF for the postirradiation colonyforming assay caused the cells to exhibit increased repair of radiation damage. These studies have proposed that radiation induces a complete cycle of an autoregulated damage/repair pathway in bovine aortic

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endothelial cells (BAEC), initiated by radiation-induced damage to cellular DNA and followed by stimulation of bFGF synthesis and its secretion into the medium. The newly synthesized bFGF stimulates the potentially lethal damage/repair (PLDR) pathway, acting via an autocrine loop leading to the recovery of cells from radiation lesions and restoration of clonogenic capacity. Furthermore, these authors have reported that in addition to bFGF, irradiation of BAEC results in an increase in PDGF mRNA. T h e fact that Raf-1 has been observed to be complexed with the PDGF receptor again emphasizes the possibility of an autocrine mechanism of signal transduction leading to increased survival after radiation exposure with Raf-1 playing a central role. The transcriptional regulation of cytokines TNF-a or IL-1 in response to radiation has also been reported (Hallahan et al., 1989; Woloschak et al., 1990). However, the radiobiological consequences of induction of these two cytokines may be different. Whereas, TNF-a is cytotoxic via such mechanisms as free radical formation (Zimmerman et al., 1989), I L 1 is reported to protect mice from lethal doses of wholebody irradiation (Oppenheim et al., 1989). More recently, a delayed synthesis of cyclin B mRNA, which encodes a cell cycle-related protein, and absence of accumulation of the cyclin B protein were observed in HeLa cells exposed to ionizing radiation during the S and G, phases, respectively (Muschel et al., 1991). Evidence that the gene(s) associated with cell cycle checkpoints may contribute to an increase in cell survival following exposure to y-radiation is also derived from other reports (Kastan et al., 1991; Kuerbitz et al., 1992). These studies demonstrate that the levels of wild-type p53 protein in hematopoietic and nonhematopoietic mammalian cells increase and decrease in temporal association with G , arrest following irradiation. More recently, the induction of gadd45 gene following ionizing irradiation has been shown to depend on a wild-type p53 phenotype (Kastan et al., 1992). Therefore, it appears that ionizing radiation affects specific molecules with important growth-related biological functions in a variety of cell types. Further investigations are necessary to provide insight into the radiobiological significance of these radiation-inducible molecular events.

VII. DNA Damage and Repair Cascade: Biochemical and Cellular Factors Ionizing radiation is known to produce a variety of free radical species, the detoxification of which may have potential implications in the phenotypic outcome of the damage caused by irradiation. A number of intracellular radioprotective molecules with a detoxification function

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22 1

have been characterized and include superoxide dismutase, catalase, glutathione (GSH) and GSH-related enzymes, protein thiols, and a number of other low-molecular-weight thiol-containing molecules (Meister and Anderson, 1983). Superoxide dismutase converts superoxide to hydrogen peroxide, whereas catalase detoxifies hydrogen peroxide to water. These two enzymes may be important in the detoxification of toxic oxygen-related species that can be produced by radiation. However, it is not clear whether high concentrations of these enzymes can protect cells from radiation damage and whether the modulations of oncogene function(s) can result in the alteration of their biochemical activities. Glutathione, a ubiquitous tripeptide, plays a critical role in several bioreductive reactions, transport, enzyme activity, protection from harmful oxidative species, and detoxification of xenobiotics (Meister and Anderson, 1983); GSH may provide radiation protection by several mechanisms including radical scavenging, restoration of damaged molecules by hydrogen donation, reduction of peroxides, and maintenance of protein thiols in the reduced state (Biaglow et al., 1983a; Clark, 1986; Mitchell and Russo, 1987; Bump and Brown, 1990).A direct correlation between intracellular GSH levels and inherent radiosensitivity has not been established (Louie et al., 1985; Mitchell et al., 1988). The depletion of cellular thiols by several reagents including diamide, N-ethylmaleimide (NEM), and DL-buthionine S,R,-sulfoximine (BSO) may render oxygenated cells more sensitive to radiation (Sinclair, 1973; Vos et al., 1976; Harris, 1979; Mitchell et al., 1983; Biaglow et al., 1983a). In this regard, the effects of BSO are of particular interest since, unlike other agents that must form a covalent bond (i.e., NEM) or oxidize GSH (i.e., diamide), BSO depletes cellular stores of GSH via a fairly specific mechanism, i.e., competitive inhibition of cysteine synthetase, a key enzyme in the biosynthesis of GSH (Griffith and Meister, 1979). Extensive cellular GSH depletion by BSO has been found to be a requirement for aerobic radiosensitization of cells (Mitchell et al., 1983; Biaglow et al., 198313; Leung et al., 1993). The mechanism of diamide-induced radiosensitization may involve oxidation of protein thiols, which are important for DNA repair (Harris, 1979).N-Ethylmaleimide possibly removes thiols from the cells or inhibits enzymes thought to be involved in the repair of lethal damage due to irradiation (Sinclair, 1973). Therefore, the possibility remains that the manipulation of the GSH and related redox systems can be effective in enhancing the cytotoxic effect of radiation and some chemotherapeutic agents in radio- and chemoresistant cells (Clark, 1986). Limited information (reviewed below) is available regarding the re-

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dox regulation of certain oncogene products via sulfhydryl modifiers (diamide, NEM), and regarding possible alterations in the intracellular levels of GSH or the GSH-related enzyme GSH S-transferase, as a result of oncogene activation o r transfection. An understanding of the role, if any, a specific oncogene(s) plays in the redox-related biochemical control mechanisms apparently elicited by y-radiation is presently lacking. A gap also exists in our knowledge of the effects of free radicals generated by y-irradiation on oncogene function(s). Several transcriptional regulatory proteins require free sulfhydryl residues for DNA binding o r transcriptional activation (Silva and Cidlowski, 1989; Levy et al., 1989). Recent studies have shown that the binding of the Jun homodimer, and the Fos plus Jun heterodimer to the AP-1 DNA binding site is inhibited by NEM. Treatment of these proteins with diamide results in their conversion to slower migrating forms most likely representing disulfide crosslinked dimers. Diamide treatment also causes inhibition of their DNA binding activities. Furthermore, a single cysteine residue in Fos and Jun was found to be important for DNA binding and that reduction was required for association with DNA (Abate et al., 1990). Sulfhydryl groups are also important in the kinase activity of p60v-src (Uehara et al., 1989). Interestingly, the last cysteine of the cysteine-finger region in the N-terminal domain of Raf-1 is critical to its dominant negative regulatory effect (Bruder et al., 1992). However, a potential role for oxidation-reduction in the control of the Raf-1 protein-serine/threonine kinase has not yet been established. It appears that a correlation between the intracellular level of GSH and activation of Raf-1 may exist. A differential modulation of intracellular GSH levels was noted as a direct response to PDGF treatment (5- 10 min) of NIH/3T3 transfectants containing different regions of transfected human raf-1 gene (Kasid et al., 1991b). Moreover, the transformation of rat liver cells with v-H-ras or v-raf is associated with expression of the multidrug resistance gene (mdr-1) and glutathione Stransferase-P, and increased resistance to cytotoxic chemicals (Burt et al., 1988). More recently, sulfhydryl modifiers, BSO and dimethylfumarate (DMF), were found to cause a graded depletion of GSH and modulation of the Raf-l-associated protein-serine/threonine kinase activity in human renal cell carcinoma-derived cells (Leung et al.,1993; U. Kasid et al.,unpublished observations). Clearly, a number of pertinent questions remain unanswered with the thiol-related biochemical regulatory mechanism(s) of Raf-1 protein kinase being one of the main issues. Another factor that may be involved in this response is the activities of DNA strand-break repair-associated enzymes topoisomerase I and 11.

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These enzymes regulate the conformation of,DNA. Besides the recently described role for these enzymes as targets for anticancer drug therapy, increased levels and activities of topoisomerases have been associated with transformed cells (Heck and Earnshaw, 1986; Crespi et al., 1988; Heck et al., 1988; Schneider et al., 1990). Moreover, it has been suggested that they may be directly involved in oncogenesis (Francis, 1987; Francis et al., 1987; Crespi et al., 1988). Evidence indicating an association between the activity of these enzymes and radiation resistance is fourfold: (a) topoisomerases are known to be activated in vitro by serinelthreonine kinases (Rottman et al., 1987; Durban et al., 1983); (6) deficiency of topoisomerase I1 has been reported to be associated with the radiosensitive phenotype of cells derived from patients with the inherited disorder ataxia telangiectasia (AT) (Mohamed et al., 1987); (c) inhibitors of topoisomerases (I and 11) potentiate ionizing radiation-induced cell killing (Mattern el ad., 1991); and (d) an elevated level of activity of topoisomerase I1 was found not only in the radiation-resistant NSF cell lines from the Li-Fraumeni family, but also in radiation-resistant NIH/3T3-transformed cell lines NIH/3T3 raf and NIH/3T3 EJ-rm (Cunningham et al., 1991). An important aspect of the radiation survival response is its regulation at the level of the cell cycle. The radiation survival experiments using synchronously dividing cell cultures have established that, in general, the cells are most sensitive at or close to mitosis, most resistant in the latter part of the S phase, sensitive in G, phase, and less sensitive in G I . In cells with a long G , phase, they are resistant in early G I , and sensitive toward the end of G I (Terasima and Tolmach, 1961; Sinclair and Morton, 1963; Whitmore et al., 1965; Sinclair, 1968). These variations in radiation sensitivity during the cell cycle have been observed despite differences in intrinsic radiosensitivity (Do value) of squamous carcinoma-derived cell lines (Quiet et al., 1991). It is noteworthy that the Chinese hamster ovary cells enriched in G, phase also reveal most sensitivity to radiation-induced mutagenesis. However, the greatest level of chemical protection from radiation-induced mutagenesis was also observed for G,-enriched populations (Grdina and Sigdestad, 1992). A number of oncogenic proteins either are known to be regulated in a cell cycle-dependent manner or have been implicated in the control of cell cycle (Hunter, 1991). The oncogenes coding for some of these proteins ( m y , r a , mos, and src) have also been implicated in intrinsic radioresistance, and some others (ras plus myc; p53) in the postirradiation changes in cellcycleorDNAsynthesis(TableI; McKenna etal., 1991;Kastan etal., 1991). It is suggested that the GI arrest and/or G , arrest induced by y-radiation allows the cells to recover from the lethal effects of radiation

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prior to cell division. Thus, alterations in this response may correlate with increased or decreased resistance to radiation. T h e RAD9 control system (or checkpoint) in yeast ensures G, arrest until the damage induced by X-irradiation is repaired, whereas mutations in RAD9 gene allow cells with DNA damage to proceed through cell division (Hartwell and Weinert, 1989). Like RAD9 defects in yeast, caffeine treatment of irradiated mammalian cells permits their entry into mitosis and decreases cell viability (Schlegel and Pardee, 1986). Using the primary rat embryo system, a significant increase in the duration of the G2 block in the radiation-resistant (rm and myc cotransfected) cells was noted compared to the relatively radiation-sensitive cells transfected with myc alone (McKenna et al., 1991). By analogy to the radiobiological characteristics of cells representing the radiosensitive genetic disorder ataxia telangiectasia, the above studies have led to the proposal that alterations in the cell division delay is one mechanism by which radioresistance is conferred on oncogene (ras and my)-transformed cells. A correlation between the expression of wild-type (wt) p53 and G, arrest, as discussed in the previous section, suggests that wt p53 may participate in the control of cell cycle progression following DNA damage (Kastan et al., 1991; Kuerbitz et al., 1992). However, these findings may be more relevant to the aspects of cellular transformation than cellular radiation resistance. As an inherited germline defect, the NSF cell lines from members of families with Li-Fraumeni syndrome have been shown to possess both a point-mutated (mt) and a wt p53 allele (Srivastava et al., 1990; Malkin el al., 1990). Nontumorigenic cell lines from one family have also been shown to express equal amounts of both the mt and the wt forms of the p53 protein (Srivastava et al., 1992). T h e NSFs from this particular Li-Fraumeni family have also been found to display a radiation-resistant phenotype (Bech-Hansen et al., 1981) (Fig. 3). This relatively radioresistant phenotype was seen not only in the cell line from an individual (V-10) homozygous for wt p53 (wt/wt), but also in the cell lines established from two individuals (VI-2 and VI-4) heterozygous with respect to the p53 point mutation (mt/wt). Furthermore, two of the radioresistant NSF cell lines that carry the mt-wt genotype actually displayed a longer lag time between exposure to X-rays and the resumption of DNA synthesis compared to an unrelated, nonradioresistant (control) skin fibroblast cell line (Paterson et al., 1985). Therefore, although the presence of one mutant p53 allele may contribute to the propensity for tumor formation by increasing genetic instability (Kuerbitz et al., 1992), it may not necessarily affect the acquisition of radiation resistance. Given the fact that some other proteins (i.e., cyclins, cdc2 ser-

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ine/threonine kinase) play a major role in the control of cell cycle (Hunt, 1989; Draetta, 1990; Hunter and Pines, 1991) in a large variety of cell types, it is conceivable that these proteins may also govern, either directly or indirectly, the radiobiological component of the oncogene function. Earlier reports have demonstrated a critical role for the mos gene product in cell cycle control by directly or indirectly stabilizing the cyclin/cdc2 complex (Roy et al., 1990). More recently, it has been suggested that the Raf-1 signaling cascade may converge in the activation of the cdc2 complex (Heidecker et al., 1992). Therefore, an alternative mechanism underlying the differential regulation of the radiation response during the cell cycle may be linked to the modulation of the cell cycle-related proteins via cytoplasmic protein-serinelthreonine protein kinases. Future studies will most likely focus on these topics for a better understanding of the connection between radiation response, the cell cycle, and oncogenes.

VIII. Modulation of Radiation Resistance: Therapeutic Implications of Oncogene Strategy As our understanding of the involvement of specific genetic factors involved in radioresistance increases, so too should our resolve to design potential therapies that can be aimed at the specific genetic o r metabolic process leading to the specific cellular manifestation. One of the most obvious long-term practical uses of the present area of research is the possibility of the radiosensitization of tumor cells by expression of a reduced level of the oncogenic protein playing a potential role in radioresistance. Since the antisense oncogene (raf 1) strategy has proven effective for the down-regulation of radiation resistance in human tumor cells (Kasid et al., 1989a), it seems logical to direct future efforts at improving upon the antisense RNA approach in order to achieve a sustained radiosensitization effect. Antisense oligonucleotides targeted at either viral o r cellular genes have been shown to be highly effective in inhibiting the expression of the targeted gene (Wickstrom, 1991; Murray, 1990; Mol and Van Derkrol, 1990). In some cases the inhibition is highly selective and the specificity reaches to that of a point mutation (Chang et al., 1991). A number of investigators have used antisense oligonucleotides as an innovative treatment strategy to block the specific genes involved in a variety of malignancies as well as in viral, inflammatory, and cardiovascular diseases. T h e development of antisense DNA technology in the last two decades has led to the present provisional approval for the first clinical trial on chronic myelogenous leukemia using an antisense strategy. In pilot

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studies, using human tumor cells, the radiosensitizing effect of the raf- 1 antisense oligonucleotides has been observed (Kasid et al., 1991a). The potential usefulness of the antisense-based experimental design may be further explored by taking advantage of the cell cycle-related variations in the radiation response and the postirradiation changes in the cell cycle. Recently, a line of transgenic mice that contains the human ruf-1 oncogene was identified as being significantly more radiation resistant than its normal, nontransgenic counterparts (K. F. Pirollo and E. H. Chang, unpublished observations). The development of such animals provides us with an in vivo model system with which to test the various potential therapies, including the antisense therapy designed to increase the therapeutic advantage of radiation-induced cytotoxicity.Thus, it is our hope that the oncogene studies may ultimately lead to the development of a specific gene-directed approach for effective radiotherapy. IX. Conclusion

There is a growing body of evidence suggesting an important role for certain oncogenes (rm,.ah cot, mos,and m y )in the regulation of cellular resistance to ionizing radiation. The observation that some of these genes demonstrate a cooperative effect toward radiation resistance is suggestive of the possibility of a selective interaction among these proteins, analogous to that involved in signal transduction leading to cell growth and proliferation or differentiation. Antisense ruf 1 cDNA transfection has been shown to cause negative regulation of radioresistance in human tumor cells, further implying that the Raf-1 protein kinase may be an important transducer of signals leading to the radiation-resistant phenotype. In addition, perturbations in the cell cycle (C, and/or G , arrest) and cell cycle-related proteins appear to be important factors contributing to cell survival. Therefore, whereas the correlation between an interaction(s) among the specific proteins and radioresistance needs to be more clearly defined, it seems likely that modifications such as serinelthreonine phosphorylation and associated transcriptional activation are critical to the radioresistant phenotype.

ACKNOWLEDGMENTS The authors acknowledge their colleagues and collaborators, especially Drs. G . Mark, A. Pfeifer, P. Ramsamooj, R. Weichselbaum, J. Mitchell, U. Rapp, D. Kaplan, and W. Anderson, for participation in the portions of studies discussed in this review; Dr. Roberta Black for helpful comments; and Elaine Miranda and Jennifer LaMontagne for excellent

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assistance in typing the manuscript. Work in the authors’ laboratories was supported by NIH Grants CA46641 and CA58984 (U.K.), CA52066 and CA45408 (A.D.),and CA45158 and CA42762 (E.C.). Additional funds were provided by National Foundation for Cancer Research Grant NFCRHUOOl (E.C.) and Uniformed Services University of the Health Sciences Grant USUHSR074DK (K.P.)

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