In1 J. Radialron Oncology i3ml Phvs Vol. Pnnted in the U.S.A. All rights reserved.

20, pp

t .oO

0360.3016/91 $3.00 Copyright 0 1991 Pergamon Press plc

295-302

l Session IIIB

KEYNOTE ADDRESS: INTEGRATION OF CYTOSTATIC AGENTS AND RADIATION THERAPY: A DIFFERENT APPROACH TO “PROLIFERATING” HUMAN TUMORS TIMOTHY

J. KINSELLA, MARK

M.D.,

A. RITTER,

MICHAEL M.D.,

N. GOULD,

PHD.

AND

PH.D.,

R. TIMOTHY

JOHN F. FOWLER,

PH.D.,

MULCAHY,

PH.D.,

D-SC.

Department of Human Oncology, University of Wisconsin Medical School, 600 Highland Ave., K4/3 12, CSC, Madison, WI 53792 Failure to achieve local and regional tumor control with radiation therapy remains a significant problem for a number of anatomic sites and can have a negative impact upon survival. There is emerging clinical and laboratory evidence that proliferation of tumor clonogens during the course of radiation treatment significantly impairs local control. Recent in situ studies suggest that as many as half of all human carcinomas have the potential to double their cell number in 5 or fewer days. Thus, cells that survive the initial treatments might rapidly repopulate a tumor, resulting in local failure. One potential clinical approach to reduce the impact of tumor cell repopulation during treatment would be to administer biological or chemical modifiers to slow or inhibit tumor proliferation. Examples of these cytostatic modifiers which are available for clinical testing now, or in the near future, include hormones, anti-hormones, growth factors, growth factor antagonists and other biologicals (e.g., interferons). Clinical alteration of the proliferative status of tumors could influence tumor control by reducing the impact of tumor cell proliferation during therapy, by modifying tumor cell radiosensitivity, or by favorably altering both. To appreciate the magnitude and the cumulative effect of these factors, newer technologies and experimental model systems need to be exploited in investigating correlations between proliferation and tumor control and between proliferative status and radiosensitivity. The design of future clinical trials using cytostatic agents and radiotherapy will rely heavily upon such basic information. Tumor proliferation, Cytostatic agents, Radiation therapy.

INTRODUCTION

idence that radiation therapy cure rates for some human tumors decrease with protraction of overall treatment time and that the basis for this is likely to be tumor repopulation during treatment (23, 32, 33, 44, 45, 50; Fowler, J. F.; Lindstrom, M. J., unpublished data, August 1990). For each doubling of tumor clonogen number, about one daily radiation fraction is wasted. Human tumors are generally perceived to grow quite slowly, with volume doubling times of many weeks or months. However, volume changes reflect the cell loss factor as well as the tumor cell proliferation rate, and cell loss factors can be as high as 80 to 99%. Thus, slow volume changes can disguise the fact that tumor clonogenic doubling times may be as brief as several days. In the context of a treatment course of conventional fractionated radiation therapy lasting 6 or 7 weeks, such potentially rapid proliferation could well compromise therapy. At least three different treatment strategies in radiation oncology are being pursued to reduce the impact of rapid tumor cell proliferation during irradiation. These include the use of altered fractionation, the use of S-phase specific

Radiation therapy has benefitted from an improving knowledge of the nature of cancer and from an evolution of technical capabilities, resulting in an improved ability to manage the cancer patient. In spite of this, the control of primary and regional disease with acceptable morbidity remains a major problem for even relatively early stage tumors in some sites and for large tumors in all sites. Eradication of primary and regional disease is, of course, a prerequisite for cure. Suit and Westgate (4 l), in an analysis of frequencies and consequences of local versus distant failures according to organ site, has concluded that significant gains in cancer survival should result if improvements can be made in loco-regional disease control for certain cancer sites, including cancer of the uterine cervix, head and neck, ovary, colorectum, lung, prostate, and bladder. The proliferation of tumor cells during treatment has been identified as a factor that might impair tumor response and local control. There is increasing clinical evReprint requests to: Timothy J. Kinsella, M.D. Acknowledgement-This work was supported in part by a Program Project Grant

Accepted for publication

from NIH (POl-CA52686-01). 295

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radiosensitizers such as the halogenated pyrimidines, and the use of biological and/or chemical modifiers to slow or inhibit tumor cell proliferation. The laboratory and clinical trials using the first two approaches have been summarized in detail recently (22, 24, 33, 44, 45, 50; Fowler, J. F.; Lindstrom, M. J., unpublished data, August 1990) and will not be discussed further. The third strategy, of reducing tumor cell proliferation by using cytostatic agents during irradiation, has not received as much laboratory or clinical interest to date. However, cytostatic agents, which inhibit cellular proliferation although not cytotoxic by themselves, are playing an increasingly important role in cancer therapy. Cytostatics such as hormones, anti-hormones, growth factors, growth factor antagonists, and other biologicals may be given during a course of radiotherapy either incidentally or by design. In this keynote address, we review the available data on cell kinetics of human cancers which emphasizes the rapid proliferation rates of many common cancers, review the available laboratory data on the radiation response of proliferating and non-proliferating tumor cells, and illustrate how the available cytostatics and radiation therapy might be used clinically in the near future. DISCUSSION Laboratory and clinical estimates of human tumor proliferation Potential doubling times (T,,) prior to treatment are thought to be of importance because they may be the best indicator of a tumor’s ability to proliferate during therapy and potentially make treatment less effective. The Twt is the time taken to double the cell population, assuming no cell\loss. The relationships of the T,, to volume doubling time (Td) and cell cycle time (T,) in human tumors are illustrated in Figure 1. Tp,, is equal to the cell cycle time divided by the growth fraction (and then multiplied by log 2). A limited number of actual measurements of proliferation rates in human tumors biopsied from patients

February 199 I, Volume 20, Number 2

prior to radiation therapy have been reported. Wilson et al. (49), using BrdUrd labelling of S-phase DNA, followed by monoclonal antibody labelling and flow cytometry, measured potential doubling times in an initial group of 26 human tumors in a variety of sites. Potential doubling times for nine head and neck tumors ranged between 3.4 and 12.3 days (mean of 6.2 d). For four rectal tumors, the range was 4.0-6.6 d; for three cervix tumors, values between 3.6 and 4.3 d were found. Ranges seen for four esophagus and three lung cancers were 3.7-9.1 (mean of 6.7) and 3.2-l 1.3 (mean of 6.7) days, respectively. More recently, Wilson, McNally, and Dische have extended their series to include 148 patients and the data are presented in Table 1 (Gray Lab Annual Report, 1989). Additionally, Begg et al. (2) have reported potential doubling times in six transitional cell carcinomas of the bladder and five head and neck squamous cell carcinomas, with mean potential doubling times of 7.4 and 7.8 days, respectively. A further 30 head and neck cancers had a mean T,,, of 4.6 days (25). Riccardi et al. (36) also performed similar analyses using the BrdUrd monoclonal technique in a number of solid and lymphoid tumors and found T,, values which averaged less than 1 week. Steel (39) as well, reviewing the older percent labeled mitoses data using tritiated thymidine, found similar values for the ranges and average potential doubling times for several different types of human tumors. The striking features of these measurements of Tw,, in human tumors prior to treatment are that, taken together, about 50% of the tumors examined had potential doubling times of 5 days or less and that, instead of a characteristic mean doubling time for each site and histology, there was large variation about the mean. The heterogeneity in kinetics implied by these limited studies suggests that individual pretreatment testing might be necessary to determine if a particular tumor falls within a rapidly proliferating class. It is obvious that much more information Table I. T,,

Type of tumor Volume Doubling

Time (T,) - - - - - - - - - - -,

All Cell Loss Factor

Potential Doubling Time (T,,)- - - - - - - - - -i

w Growth Fraction

i

cell Cycle Time (T,) __ __ __ __ ___ __ __:

Fig. 1. Inter-relationship of volume doubling time (Td), potential doubling time (T,,), and cell cycle time (T,).

Lung Columel. Esoph. Rectum Mal. mel. Tongue Cervix Larynx Mouth, alveolus and cheek Tonsil

from FCM and in situ BrdUrd-

N

Range of T,, (days)

Median (days)

I48 patients Proportion 10% after five fractions of a 20-fraction course of treatment was an absolute predictor of failure. These investigators also found that an accelerated fractionation scheme was more effective in depressing the labeling index. There are only scant human data on tumor cell kinetics during treatment. Silvestrini et al. (37) investigated cancers of the oral cavity and found that 270% drop in labeling index after five 2 Gy fractions was highly predictive of ultimate complete local response and correlated with improved disease-free survival. Such analyses of tumor cell kinetics during treatment are complicated by the inclusion of lethally irradiated, but intact, cells in the assay. At present, many experts argue that it is not possible technically to measure T, in patients during therapy with the flow cytometry methodology using BrdUrd/IdUrd specific monoclonal antibodies because of the presence of these intact, non-viable cells. Newer techniques using cell proliferation markers such as Ki-67, PCNA (cyclin), and ribonucleotide reductase (MI) alone or combined with the BrdUrd/IdUrd monoclonal antibodies are subject to some of the same limitations, but hold promise in providing supplementary kinetic information. In summary, there is increasing laboratory and clinical evidence that the proliferation rates of many human tumors are quite fast (in the order of several days) and that tumor cell proliferation during treatment may increase the risk of local failure following irradiation. At present, the development of specific anti-BrdUrd and anti-IdUrd monoclonal antibodies provides the necessary tools for the clinical investigator to measure human tumor cell kinetics in various anatomic sites in patients before treatment. The potential clinical relevance of these pretreatment measurements of T,, was highlighted recently by Moonen et al. (25). As part of a randomized prospective EORTC trial of conventional fractionation versus accelerated fractionation in patients with head and neck cancer, there was a trend toward improved local control favoring accelerated hyperfractionation in patients whose pretreatment T, was ~4.6 days. Although interesting, these data need longer follow-up and confirmation with larger numbers of patients. In vitro/in vivo radiation response of proliferating vs. non-proliferating (“quiescetzt’7 cells In vivo tumors contain both proliferating and non-dividing (quiescent) cell populations. The ratio of these populations can contribute, in part, to the heterogeneous

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response of tumors to ionizing radiation. Published data examining the relative radiosensitivities of quiescent versus proliferating cells are not consistent. Starting in the early 1970’s, several investigators pointed out the importance of the quiescent cell population in growing tumors and began investigating its relative radiation sensitivity. Barendsen and colleagues used a transplantable rat rhabdomyosarcoma to investigate this question. They reported that the quiescent tumor cell population was more radioresistant than the proliferating population (1, 17). These findings were independently corroborated in animal tumors in vivo by both Kallman and colleagues (2 1) and Goldfeder and colleagues (34). Although these results were intriguing, they were difficult to interpret, since the tumor quiescent cell population often overlaps with the radioresistant hypoxic cell population (47). To overcome the confounding influence of a hypoxic cell population while still using an in vivo model, Gould and Clifton began to investigate this problem in normal tissue. In 1978, they reported that quiescent mammary cells from virgin rats had the same radiosensitivity as rapidly proliferating, hormonally stimulated rat mammary cells (15). This result with normal epithelial cells contrasted those reported for in vivo tumors. In the early 1980’s, it became apparent that the question of the radiosensitivity of the quiescent cell population should be addressed in more readily controllable in vitro models. Appropriate models for these studies include both monolayer cultures as well as multicellular spheroids. Among the best characterized in vitro monolayer models for the radiobiological study of quiescent cells are the murine mammary carcinoma models developed by Wallen and colleagues (47). It was clearly demonstrated that these cell lines could efficiently establish a uniform quiescent cell population after nutrient deprivation (7 day, unfed plateau cultures). Using this model, it was demonstrated that quiescent cells of two different cell lines were more radiosensitive than their proliferating counterparts (47). In contrast to these findings, Hlatky et al. using nuttientdeprived 9L tumor cell model, found that these cells in their quiescent state were more radioresistant than their exponentially growing counterparts ( 18). However, results with the V-79 spheroid model seem to parallel the former finding. Durand (9) showed that the quiescent interior cells of multicellular spheroids were more radiosensitive than the exterior proliferating cells, after any interior hypoxic effects were accounted for. Although it is quite reasonably argued that these nutritionally-based systems may adequately model tumor populations located beyond the diffusion limits of critical nutrients, their relevance to the radiation responsiveness of cells out of cycle as a result of growth factor alterations or hormonal modulations is questionable. Inconsistencies among the above described in vitro models may be either cell line dependent or due to differences in the methods used to induce a quiescent subpopulation. Methodological

February 199 I, Volume 20, Number 2

variation arises in part from the non-specific methods used to induce quiescence (i.e., nutrient deprivation; position in a spheroid). This lack of specificity also limits approaches that may be used for investigations into fundamental processes. To circumvent problems associated with this nonspecificity, we sought to develop models that induced quiescence by more defined alterations in culture medium. Several models have been established in our laboratories. In the first, a normal rat thyroid cell line was used. This cell line is dependent on thyroid stimulating hormone (TSH) for proliferation. Removal of TSH from the medium induces quiescence. In this model, proliferating and quiescent thyroid cells had identical radiosensitivities (5). These results are in agreement with our early in vivo rat mammary data (15). In contrast to these results, other studies in our laboratory showed that when B-interferon was used to inhibit proliferation in a human bronchogenic cell line, the cells so treated became more radiosensitive ( 16). In vitro radiation sensitization by various interferons of other tumor cell lines has also been reported (7,8,26). Although these latter findings are intriguing, they are preliminary and require detailed follow-up. More recently, we have developed a series of human primary cell models that are useful to explore the radiosensitivity associated with the quiescent state. Human normal and malignant breast and prostate epithelial cells are grown in serum-free medium. A non-proliferative state can be induced by growth factor (GF) deprivation, that is, by removal of epidermal growth factor (EGF) or pituitary extract (PE). In all four cell types, we find that quiescent cells are generally more radiosensitive than their proliferating counterparts. These results contrast with those obtained with TSH-deprived thyroid cells. The possibility exists that GF deprivation deprives the cells of more than just a mitogenic signal, while TSH may be acting primarily as a mitogen. This possibility is reinforced by our finding that when mammary cells are made nonproliferative by contact inhibition, their cell cycle distributions are identical to those made non-proliferative by GF deprivation. However, although GF deprivation increases radiosensitivity when compared to proliferating cells, contact inhibition does not. These effects on the in vitro radiosensitivity of a primary breast cancer culture are illustrated in Figure 2. Clearly, these intriguing preliminary observations of altered radiosensitivity in human breast cancer with growth factor deprivation need further study. With the increasingly common addition of cytostatic agents, such as tamoxifen (TAM) and interferons, to conventional cytotoxic therapy, including radiation, the potential significance of their role in modifying radiation responsiveness and ultimately tumor response also increases. While investigations of the effects of certain growth modulating agents on radiation response have been proposed because of potential advantages they might

Tumor

cell proliferation

INFILTRATING DUCTAL CARCINOMA 3556 0 Exponential W Contact Inhibited Before 7 A Growth Factor Deprived Before 7 A Growth Factor Deprived Before k After 7 •I Contact Inhibited Before & After 7

1 .OOOE-5 0

300

600

DOSE

900

1200

(cGy)

Fig. 2. In vitro radiation dose-cell survival curves for a primary explant culture of human breast ductal carcinoma. Culture conditions are illustrated on figure. Growth factor deprived is defined by removal of epidermal growth factor from culture media for 24 hr prior to irradiation (7).

offer when combined with radiation in future treatment strategies, combinations including radiotherapy and TAM are already a clinical reality and currently the focal point

of considerable concern. In specific clinical circumstances, such as definitive breast irradiation in a post-menopausal, estrogen receptor positive (ER+) patient, as well as in numerous ongoing breast cancer clinical trials, the initiation of TAM therapy precedes or coincides with tumor irradiation. The advisability of such combinations has been questioned in light of the demonstrated cytostatic effect of TAM on human breast cancer cells in vitro and the possible radioresistance of non-cycling cells. In apparent confirmation of this possibility, Wazer et al. (48) recently reported that the radiation responsiveness of MCF-7 breast cancer cells in vitro was significantly reduced by TAM exposure, heightening concern over possible antagonistic effects of TAM and radiation. Since the resultant increase in survival was predominately attributable to an increased shoulder on the survival curve (48), this effect may be particularly relevant to the small dosesper-fraction used in clinical radiotherapy. Preliminary data from our labs using MCF-7 cells show no change in radiation response. Whether or not the effect of TAM therapy on in vitro radiation response can be extrapolated to the in vivo situation may be dependent upon TAM’s ability to reduce tumor proliferation in vivo. Although data demonstrating a cytostatic effect of TAM in vitro are legion (27,28,42), similar data for the in vivo situation are surprisingly limited and contradictory. Osborne and co-workers (29) reported that TAM markedly reduced the mitotic index of MCF-7 xenografts whereas in the only other published report, Brunner et al. (6) failed to observe any differences between the proliferation kinetics for control and TAMtreated MCF-7 xenografts. Considering the potential importance of the anti-proliferative effect of TAM on the radioprotection observed in TAM-treated MCF-7 cells in

0 T. J. KINSELLA ef al.

299

vitro, the potential discrepancy between mechanisms of TAM action in vitro and in vivo, and the lack of agreement on the effect of TAM on cell proliferation in vivo, we feel that the potential impact of TAM therapy on the radiation response of breast tumors would best be addressed in human tumor xenograft models. The in vivo interaction of TAM and related anti-estrogens with radiation is currently under study in our labs using the well-characterized MCF7 human breast cancer xenograft model ( 13, 14). In summary, it is well established that pre- and postirradiation environmental factors can profoundly influence the response of mammalian cells to radiation exposure. Although the effects of several such factors have been examined experimentally, a considerable amount of interest has recently centered on the effect of proliferative status on radiation response, with an emphasis on determining the role that cellular quiescence plays in the response of tumors to therapeutic irradiation. Research in this area has been greatly advanced by the development of experimental systems to model quiescent populations reproducibly and to identify quantifiable parameters characteristic of quiescent cells. Unfortunately, conclusions regarding the relative sensitivity of quiescent populations have frequently been conflicting, a fact which may reflect the complex nature of the accompanying cellular physiology or alternatively may simply be attributable to differences in the techniques employed to induce quiescence. It is important for the Radiation Oncology community to use the available in vitro and in vivo human cell model systems to begin to understand the effects of these various types of cytostatic agents on the radiation response of human tumors. Based on these experimental results, clinical protocols can then be developed in a more scientific fashion. Potential clinical integration of cytostatics and radiation therapy A major concern in combining a cytostatic agent with radiation therapy is whether any potential gain in slowing tumor cell proliferation might be lost if there is a resulting reduction in radiation sensitivity. Information on tumor cell kinetics prior to and during therapy, as well as some measure of intrinsic cellular radiosensitivity, will be important laboratory endpoints to monitor in any future clinical trial. Additionally, similar types of measurements of dose-limiting normal tissues will need to be performed. As discussed previously, these types of biological endpoints (i.e., tumor cell kinetics using anti-BUdR/IUdR monoclonal antibodies and/or proliferation markers such as Ki 67; establishing primary tumor explants for in vitro radiation studies) may be ready for clinical testing in the near future. Given the ability to carry out these biological studies in an individual patient, it will also be important to establish some general guidelines to predict for a favorable clinical outcome. As an example, we have extrapolated

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% -

106

_ Calc far 30f Y 2Gy.

. lo8 cells tumor

burden

t Teff=3d

4.5d

xl xl.5 FACTOR OF

6d

12d

24d

d

48d’

OJ

x16 CfI x2 x4 x8 INHIBITION OF PROLIFERATION

Fig. 3. Linear quadratic modeling of inhibition of proliferation in breast cancer during conventional fractionated irradiation (2 Gy X 30 fractions). The calculations are based on the in vitro survival data from Fig. 2. As proliferation is inhibited progressively more (from left to right in the figure), the surviving cell burden becomes smaller.

the survival data on the human breast cancer explant from Figure 1 and plotted the impact of cell burden at the end of 30 fractions of 2 Gy versus the effective doubling time (T& (Fig. 3). If the T~rr (=T,,) is less than 5 days, then there is a low likelihood of ultimate control (arbitrarily defined as >90% cure). However, if one can inhibit proliferation by a factor of two or more, then improved tumor control results. For this breast tumor, the potential use of growth factor inhibitors such as suramin (3,40), which is now in clinical Phase l/II testing, or possibly mammastatin, a newly discovered specific inhibitor breast tumor proliferation (lo), might be tested clinically with breast irradiation in the future. A more clinically relevant example of combining a cytostatic and radiation therapy in breast cancer involves the present day use of tamoxifen. In conventional treatment settings, TAM therapy is initiated shortly after surgery and is frequently continued during and after adjuvant therapy including systemic chemotherapy and radiation treatment. The ubiquitous use of TAM in breast cancer patients and its ability to inhibit proliferation of breast cancer cells (27, 43) have raised concerns about the use

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of conventional tumoricidal therapies in conjunction with this known tumoristatic agent. This concern is supported by experimental studies that have identified antagonistic interactions between TAM and certain chemotherapeutic agents commonly used in breast cancer therapy (12, 20, 30). While TAM-induced inhibition of proliferation is obviously contributory to diminished responsiveness to S-phase specific chemotherapeutic drugs, other mechanisms, including inhibition of drug transport, have also been implicated ( 12,20,30). In a study recently completed by the National Surgical Adjuvant Breast Program (NSABP), the use of TAM with melphalan and fluorouracil was found to have an adverse effect on certain patient subpopulations (11). Collectively, the available data from experimental and clinical experience suggest prudence when administering TAM with certain chemotherapeutic drugs or treatment regimens in order to avoid antagonistic interactions. In light of this antagonism, it is reasonable to query: does the anti-proliferative effect of TAM exert a similar antagonistic influence on the radiation responsiveness of human breast cancer cells? If so, the extremely common practice of administering TAM prior to and during radiation therapy might prove counterproductive. Unfortunately, only preliminary evidence addressing this practical question is currently available (3 1). Although the deliberate slowing of proliferation of malignant cells appears at first to be advantageous, there are reasons, set out above, why this might not be so. The most obvious reason concerns S-phase-specific chemotherapeutic agents, because there will be a smaller proportion of malignant cells in S if their proliferation is inhibited. The second reason concerns the different radiosensitivities of cell in long G, or GZ phases. Here the evidence is contradictory, as discussed above. Further, if cells are not passing through the stages of known radiosensitivity, M and the Gi :S interface, it may be more difficult for repeated treatments to kill them. On the other hand, if tumor cells could be induced to concentrate in a radiosensitive phase such as Gz, then eliminating them would be easier. Obviously, there is scope for much research in this area of modified proliferation rates, both in laboratory and, as results are obtained, in the clinic.

REFERENCES Barendsen, G. W.; Roelse, H.; Hermens, A. F.; Madhuizen, H. T.; van Peperzeel, H. A.; Rutgers, D. H. Clonogenic capacity of proliferating and nonproliferating cells of transplantable rat rhabdomyosarcoma in relation to its radiosensitivity. JNCI 5 1: 152 1-1526; 1973. 2. Begg, A. C.; Moonen, L.; Hofland, 1.; Dessing, M.; Bartelink, H. Human tumour cell kinetics using a monoclonal antibody against iododeoxyuridine: intratumour sampling variations. Radiother. Oncol. 11(4):337-347; 1988. 3. Betsholtz, C.; Johnsson, A.; Heldin, C.-H.; Westermark, B. Efficient reversion of simian sarcoma virus-transformation

and inhibition of growth factor-induced mitogenesis by suramin. Proc. Natl. Acad. Sci. USA 83:6440-6444; 1986. Braunschweiger, P. G.; Schenken, L. L.; Schiffer, L. M. The cytokinetic basis for the design of efficacious radiotherapy protocols. Int. J. Radiat. Oncol. Biol. Phys. 5:37-47; 1979. Brosing, J. W.; Giese, W. L.; Mulcahy, R. T. Lack of a differential radiation response for proliferative and nonproliferative rat thyroid cells (FRTL-5) in vitro. Int. J. Radiat. Oncol. Biol. Phys. 16: 15 1 l- 15 17; 1989. Brunner, N.; Bronzert, D.; Vindelov, L. L.; Rygaard, K.; Spang-Thomsen, M.; Lippman, M. E. Effect on growth and

Tumor cell proliferation 0 T. J. cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice. Cancer Res. 49: 15 I 5- 1520; 1989. 7. Chang, A. Y.; Keng, P. C. Potentiation of radiation cytotoxicity by recombinant interferons, a phenomenon associated with increased blockage at the G2-M phase of the cell cycle. Cancer Res. 47:4338-4341; 1987. 8. Dritschilo, A.; Mossman, K.; Gray, M.; Sreevalsan, T. Potentiation of radiation injury by interferon. Am. J. Chn. Oncol. 5:79-82; 1982. 9. Durand, R. E. Oxygen enhancement ratio in V79 spheroids. Radiat. Res. 96:322-334; 1983. 10. Ervin, P. R.. Jr.; Kaminski, M. S.; Cody, R. L.; Wicha, M. S. Production of mammastatin, a tissue-specific growth inhibitor, by normal human mammary cells. Science 244: 1585-1587; 1989. 1 1. Fisher, B.; Redmond, C.; Brown, A.; Fisher, E. R.; Wolmark, N.; Bowman. D.; Plotkin, D.; Walter, J.; Bornstein, R.; Legault-Poisson, S.; Saffer, E. A.; other NSABP Investigators. Adjuvant chemotherapy with and without tamoxifen in the treatment of primary breast cancer: five year results from the National Surgical Adjuvant Breast and Bowel Project Trial. J. Clin. Oncol. 4:459-47 I; 1986. 12. Goldenberg, G. J.; Froese, E. K. Antagonism of the cytocidal activity and uptake of melphalan in human breast cancer cells in vitro. Biochem. Pharmacol. 34:763-770; 1985. 13. Gottardis, M. M.; Jiang, S.-Y.; Jeng, M.-H.; Jordan, V. C. Inhibition of tamoxifen stimulated growth of an MCG-7 tumor variant in athymic mice by novel steroidal antiestrogesn. Cancer Res. 49:4090-4093; 1989. 14. Gottardis, M. M.; Wagner, R. J.; Borden, E. C.; Jordan, V. C. Differential ability of antiestrogens to stimulate breast cancer cell (MCF-7) growth in vivo and in vitro. Cancer Res. 4914765-4769; 1989. 15. Gould, M. N.; Clifton, K. H. The survival of rat mammary gland cells following irradiation in vivo under different endocrinological conditions. Int. J. Radiat. Oncol. Biol. Phys. 4:629-632; 1978. 16. Gould, M. N.; Kakria, R.; Borden, E. C.; Olson, S. Radiosensitization of human bronchogenic carcinoma cells by interferon beta. J. Interferon Res. 4: 123-128; 1984. 17. Hermens, A. F.; Barendsen, G. W. The proliferative status and clonogenic capacity of tumor cells in a transplantable rhabdomyocarcoma of the rat before and after irradiation with 800 rad ofx-rays. Cell. Tissue Kinet. 11:83-100; 1978. 18. Hlatky, L.; Alpen, E. L.; Yee, M. K. Differences in the xray sensitivities of cells in different regions of the sandwich, a diffusion-limited system for cell growth. Radiat. Res. 108: 62-73; 1986. 19. Hliniak, A.; Maciejewski, B.; Trott, K. R. The influence of the number of fractions, overall treatment time and field size on the local control of cancer of the skin. Br. J. Radiol. 56:596-598; 1983. 20. Hug, V.; Hortobagyi, G. N.; Drewinko, B. Tamoxifen-citrate counteracts the antitumor effects of cytotoxic drugs in vitro. J. Clin. Oncol. 3:1672-1677; 1985. 2 I. Kallman, R. F.; Combs, C. A.; Franko, A. J.; Furlong, B. M.; Kelley, S. D.; Kemper, H. L.; Miller, R. G., Rapacchietta, D.; Schoenfeld, D.; Takahashi, M. In: Meyn, R. E., Withers, H. R., eds. Radiation biology in cancer research. New York: Raven Press; 1980:397-414. 22. Kinsella, T. J.; Mitchell, J. B.; Russo, A.; Morstyn, G.; Glatstein, E. The use of halogenated thymidine analogs as clinical radiosensitizers: rationale, current status and future prospects: non-hypoxic cell sensitizers. Int. J. Radiat. Oncol. Biol. Phys. 10: 1399- 1406; 1984. 23. Maciejewski, B.; Preuss-Bayer, G.; Trott, K. R. The influence

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