[CANCER RESEARCH 53, 5676-5679, December l, 1993] Therapeutic Tumor-specific Cell Cycle Block Induced by Methionine Starvation in Vivo 1 Huiyan Guo, Valeryi K. Lishko, Hector Herrera, Ami Groce, Tetsuro Kubota, and Robert M. Hoffman 2 Anticancer, Inc., 7917 Ostrow Street, San Diego, California 92111 [11. G., V K. L., 14. H., A. G., R. M. H.]; Laboratory of Cancer Biology, University of California, San Diego, La Jolla, California 92093-0609 JR. M. H.]; and Department of Surgery, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku Ku, Tokyo 160, Japan [T. K.] ABSTRACT The ability to induce a specific cell cycle block selectively in the tumor could have many uses in chemotherapy. In the present study we have achieved this goal of inducing a tumor-specific cell cycle block in vivo by depriving Yoshida sarcoma-bearing nude mice of dietary methionine. Fur- ther, we demonstrate that methionine depletion also causes the tumor to eventually regress. The antitumor effect of methionine depletion resulted in the extended survival of the tumor-bearing mice. The mice on the methionine-deprived diets maintained their body weight for the time pe- riod studied, indicating that tumor regression was not a function of body weight loss. The data reported here support future experiments utilizing methionine depletion as a target for tumor-selective cell cycle-dependent therapy. INTRODUCTION There has been a continuing search for agents that can selectively arrest tumor cells, in particular with a specific cell cycle block occur- ring only in the tumor. Such a tumor-specific cell cycle block could possibly be exploited by additional cell cycle-specific chemotherapy. In this light, in vitro studies have suggested that targeting the excessive methionine dependence of tumors may exert tumor-selec- tive efficacy via a tumor-specific cell cycle block (1-13). Under conditions of a limiting methionine source in vitro, methionine-de- pendent tumor cell lines arrest in the late-S/G2 phase in the cell cycle (8, 9), which we have termed the MDCCB. 3 In vitro, the combination of methionine starvation and cell cycle-specific chemotherapy used in cocultures of tumor and normal cells eliminated the tumor cells while allowing the normal cells to flourish (13). We have recently demonstrated that fresh human tumors grown in vitro show methionine dependence by measuring the MDCCB (9). Normal cells and tissues tested are methionine independent and grow after methionine is replaced by homocysteine (1, 3, 8). Methionine dependence may be due to overutilization of methio- nine for transmethylation reactions resulting in low free-methionine pools and low S-adenosylmethionine/S-adenosylhomocysteine ratios, thereby blocking cell division under conditions of a limiting methio- nine source (11-13). A number of investigators have attempted to exploit the methionine dependence of tumors for therapeutic effects in vivo. Breillout et al. (14) found for the RMS-J1 rat rhabdomyosarcoma tumor that a me- thionine-depleted diet lowered the metastatic potential of the tumor while not having significant effects on local tumor growth in rats. Goseki et al. (15) found that a methionine-free TPN mixture for rats bearing the Yoshida sarcoma extended the survival of the rats and slowed tumor growth of the rats, especially with the use of doxoru- Received 7/12/93; accepted 9/27/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by National Cancer Institute SBIR Grant R44CA43444 and National Cancer Institute Grant R01CA27564. z To whom requests for reprints should be addressed. 3 The abbreviations used are: MDCCB, methionine-dependent cell cycle block; TPN, total parenteral nutrition; TETW, total estimated tumor weight. bicin. Kreis observed that methioninase slowed the growth of the W-256 rat carcino-sarcoma in rats (16). We demonstrate in this report that the Yoshida tumor growing in nude mice can be induced by a methionine-free diet to have a MDCCB, indicating that a tumor-selective cell cycle block can indeed be achieved in vivo. We also report here that the Yoshida tumor in the nude mice can actually regress with prolonged dietary methionine starvation, resulting in an extended survival period of the mice. MATERIALS AND METHODS Mice. Four- to 5-week-old outbred nu+/nu+ mice were divided randomly into 2 groups. The mice were bred and housed in a high-efficiency particulate air filtered barrier room under NIH guidelines. A total of 21 mice were used in this study. Cells. A suspension of Yoshida sarcoma containing 1 • 107 cells previ- ously grown in Eagle's minimum essential medium with 10% fetal calf serum was injected in each mouse at the axillary and inguinal site. Diets. Defined diets with and without methionine were obtained from Tek- lad (Madison, WI). The contents of the diets are listed in Table 1. The me- thionine-free diet was depleted of homocysteine and choline to allow extensive depletion of methionine in the animal. Mice in one group were fed a methio- nine-containing diet, while mice in the other group were fed a methionine-free diet. Mice were given equal measured amounts of each diet. The defined diets were started on day 2 after the injection of Yoshida sarcoma ceils. The mice on the methionine-free diets were housed separately from mice on the methionine- containing diets. Both groups of animals ate all the food supplied to them. Tumor Weight and Carcass Weight Measurements. Measurements were made at both injection sites, axillary and inguinal. The lengths of the major and minor axes were measured with calipers. The growth of the tumors was evaluated every 3 days. Estimated tumor weight (Table 2) was calculated by the equation of Goseki et al. (15). TETW (see Fig. 2B) was calculated by a modification of the equation of Goseki et al. (15), as follows: (A + B ) TETW (rag) - 4 where TETW (mg) is the combined tumor weight of both the axillary and inguinal site, A is (length of the axillary major axis) 2 x (length of the axillary minor axis), and B is (length of the inguinal major axis) e x (length of the inguinal minor axis). Carcass weight was calculated by subtracting the TETW from the measured body weight. DNA Staining. Slides containing histological 3-~m sections of the s.c. grown tumor and normal colon and liver tissue taken immediately after death from animals 6--9 weeks old were incubated in preheated 1 Nhydrochloric acid at 60~ for 20 rain. The slides were then rinsed with distilled water 6 times to remove excess acid, stained with Schiff's reagent for 20 rain at room tempera- ture, treated with 2 successive changes of freshly prepared sulfurous acid rinse, and washed in running tap water for 5 min. After dehydration, slides were coverslipped for DNA analysis. Cell Cycle Position Determination by DNA Measurement. A Cambridge Instruments Quantimet-520 system was used to determine the DNA content of cell nuclei by measuring integrated absorbance of each cell (9); 500 cells were measured per slide. The integrated absorbance was directly proportional to DNA content. The G1 reference peak in the tissues of the control animals was determined as the first major peak in the histogram. Statistical Analysis. Based on our recent report (9), the MDCCB value was calculated by the equation G1/total cells (for methionine-containing diet)/Gt/ 5676 Research. on January 20, 2022. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from