Proc. Nati. Acad. Sci. USA Vol. 76, No. 8, pp. 3937-3941, August 1979 Cell Biology Loss of epidermal growth factor requirement and malignant transformation (serum-free medium/transformed phenotypes/growth control/diploid Chinese hamster embryo fibroblasts) PAUL V. CHERINGTON*t, BARBARA L. SMITH*t, AND ARTHUR B. PARDEE*t§ *Laboratory of Tumor Biology, Sidney Farber- Institute, 44 Binney Street, Boston, Massachusetts 02115; tDepartment of Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115; and tDepartment of Microbiology and Molecular Genetics, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115 Contributed by Arthur B. Pardee, May 18, 1979

ABSTRACT Serum provides growth factors that regulate factors whose role is modified by different modes of transfor- and limit the growth of normal cells in tissue culture. Animal mation. We specifically have measured growth of monolayer cells that are malignantly transformed usually exhibit dimin- cultures of Syrian and Chinese hamster embryo fibroblasts ished serum requirements for growth in culture. We have used by different means. a defined, serum-free medium to determine which of these transformed growth factors becomes dispensable for the growth of trans- formed Syrian and Chinese hamster fibroblast cells. The me- MATERIALS AND METHODS dium's four growth factors-epidermal growth factor (EGF), insulin, fibroblast growth factor, and transferrin-were added Cell Lines. Syrian baby hamster kidney fibroblasts (BHK- or omitted as desired. A decreased requirement for EGF was 21), clone 13 (15) from the American Type Culture Collection most closely related to tumorigenicity of chemically (ethyl were obtained from Richard Hoover (Department of , methanesulfonate) transformed cells in nude mice. All lines Harvard Medical School, Boston, MA). Polyoma virus-trans- examined retained their requirement for transferrin, which is formed BHK-21 cells, clone Ji (PyBHK) (1) were obtained from needed throughout the growth cycle, in contrast to the other Michael Stoker (Imperial Cancer Research Fund Laboratories, factors, which are needed primarily in GI phase. Lines that had lost their EGF requirement but had retained their insulin re- London). Clone MT1-C2 was derived from a tumor formed quirement were arrested in GI by insulin deficiency, indicating after subcutaneous injection of 106 BHK-21 cells into a nude that their growth control system remained. Mutagenesis with mouse. The diploid Chinese hamster embryo fibroblast clones ethyl methanesulfonate can also create requirements of the (CHEF/18 and CHEF/16) were isolated from a culture of the transformed cells for unknown factors in serum. We conclude same embryo (16). They are distinct in that the CHEF/16 clone that an initial step that reduces the serum requirement in cul- forms colonies in low serum, is tumorigenic in nude mice, and ture, and in tumorigenesis, is relaxation of the growth-regulatory forms colonies in methylcellulose, indicating no anchorage function of EGF. dependence for growth. Stocks of these cell lines were obtained Serum is a source of growth factors for cells; some of these from Ruth Sager (Sidney Farber Cancer Institute, Boston, MA). factors are known and others are not yet identified. A decreased The following lines were selected (B. L. Smith and R. Sager, requirement for serum in culture media is generally found after unpublished) after nine doublings of a CHEF/18 population cells are malignantly transformed (1-7). This change appears mutagenized (16% survival) with ethyl methanesulfonate at 200 to be one of the first among many phenotypic alterations ob- ,ug/rnl; 21-2 was a clone derived from a single round of selection served after transformation. Some of the serum growth factors in methylcellulose and is weakly tumorigenic (2 tumors in 12 have been chemically identified; transformation has been re- nude mice); 21-2-M3 was a clone derived from 21-2 after an ported to specifically decrease the cell growth requirements additional selection in methylcellulose and is also tumorigenic for some of these growth factors (8-11). (2 tumors in 4 mice); T30-4 was a subclone of a tumor arising DNA tumor viruses transform cells so that only a minimal after subcutaneous injection of 107 21-2-M3 cells into a nude serum requirement remains. Transformation with chemicals, mouse. These chemically transformed lines form colonies in low with RNA tumor viruses, or "spontaneously" is qualitatively serum concentrations. All Chinese hamster lines in this study different, and only moderately decreases the requirement for were diploid. serum (4-7). We propose that transformation by chemicals, Stocks of BHK-21, MT1-C2, and PyBHK were maintained which probably initially mutate one or a few genes (12) or by in Dulbecco's modification of Eagle's medium (DME; Flow RNA viruses that may introduce extra copies of one cellular Laboratories, Rockville, MD) supplemented with 10% calf gene (13), could initially modify the requirement for one serum (Flow Laboratories), glutamine (4 mM), penicillin (100 growth-limiting factor. Later changes of these cells could di- units/ml), and streptomycin (100 ,ug/ml). Stocks of CHEF/18 minish other growth factor requirements. In contrast, trans- and CHEF/16 and the CHEF/18-derived cell lines were formation by DNA tumor viruses is proposed to bypass an entire maintained in the alpha modification of Eagle's medium (alpha; growth factor-dependent control system, and thereby eliminate Flow Laboratories) supplemented as with DME except that calf requirements for most factors. serum was replaced by fetal calf serum (Flow Laboratories). In this study we have utilized a serum-free medium sup- All cultures were incubated at 370C in a water-saturated at- plemented only with epidermal growth factor (EGF), insulin, mosphere containing 10% CO2. Both the DME and the alpha fibroblast growth factor (FGF), and transferrin (14). By omit- media contained 3.7 g of NaHCO3/liter. Fresh cells were ob- ting single or multiple supplements we are able to identify tained from frozen stocks every 4-6 weeks. Each stock was The publication costs of this article were defrayed in part by page Abbreviations: FGF, fibroblast growth factor; EGF, epidermal growth charge payment. This article must therefore be hereby marked "ad- factor; 4F, FGF/EGF/insulin/transferrin; DME, Dulbecco's modi- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate fication of Eagle's medium. this fact. § To whom reprint requests should be addressed. 3937 Downloaded by guest on September 26, 2021 3938 Cell Biology: Cherington et al. Proc. Natl. Acad. Sci. USA 76 (1979) determined to be free of contamination by using was nearly as effective in supporting the growth of BHK-21 and the ratio of [3H]uridine and [3H]uracil incorporation (17). PyBHK fibroblasts as media supplemented with 10% calf Serum-Free Growth Media. Modified Eagle's medium serum. When exponentially growing BHK-21 fibroblasts were (alpha) and F12 were mixed 1:1 and supplemented with glu- shifted from 10% serum into the defined medium containing tamine (4 mM), penicillin (100 units/ml), and streptomycin 4F, they continued to grow for several days. Their growth rate (100 Ag/ml). This medium is referred to as alpha/F12. The and their final density were slightly reduced (to 65-70%), as defined medium (alpha/F12 + 4F) is based upon one suggested compared to their growth in 10% serum (Fig. 1). Removal of by T. Maciag (personal communication) containing FGF, EGF, FGF, EGF, and insulin resulted in less than a doubling in cell insulin, and transferrin. FGF and EGF were each added at 10 number and an arrest of the population mainly in G1 (see Fig. ng/ml. Insulin (Sigma; bovine pancreas) and transferrin (U.S. 6). PyBHK cells, like BHK-21, grew slower in 4F-supplemented Biochemical Corp., Cleveland, OH; human plasma) were added media than in serum-supplemented media, although their at 10 jug/ml and 5 jtg/ml, respectively. The FGF and EGF overall growth rate was faster in both. Thus the serum-derived preparations were the generous gift of Tom Maciag (Collabo- factors that were absent from the 4F-supplemented medium rative Research, Waltham, MA). We found that BHK cells grew and were required by BHK-21 fibroblasts appeared to be re- best when the medium was supplemented with 2.5 AtM FeSO4, quired by PyBHK cells as well. which was added to all serum-free media unless specified Although BHK-21 cell growth has previously been reported otherwise. All defined media supplements were stored at in defined media (14), no such system for the growth of -20'C. FGF, EGF, and transferrin were dissolved in phos- nontumorigenic hamster cells has as yet been reported (BHK-21 phate-buffered saline/10% (vol/vol) glycerol. Insulin was is tumorigenic in nude mice). Fig 2 shows that the nontumor- prepared in phosphate-buffered saline/10% glycerol plus 0.1 igenic CHEF/18 and tumorigenic CHEF/16 diploid Chinese M NaOH sufficient to dissolve the powder. hamster embryo fibroblast clones described by Sager and Kovac Cell Growth Measurements in Serum-Free Media. Each (16) grew in alpha/F12 + 4F. The behavior of these two clones cell line to be tested was plated in alpha/F12 + 10% serum at in this medium was analogous to the behavior of BHK-21 and 3 X 103 cells per cm2 in 2 ml per 35-mm plate or 5 ml per PyBHK in that the nontransformed CHEF/18 cells grew at a 60-mm plate and incubated for one day at 370C. Each plate was slower rate than the tumorigenic, more transformed, CHEF/16 then thoroughly washed with alpha/F12 prior to feeding with cells. The large difference in growth rate and saturation density serum-free medium. At the appropriate intervals samples were of CHEF/18 in alpha/F12 supplemented with either 10% trypsinized, suspended in Hanks' balanced salts plus 0.5% serum or 4F indicated requirements for additional factors not , and counted immediately with a Coulter present in 4F. CHEF/16 did not need these factors. Counter, model B. Some of the cell lines disintegrated upon trypsinization after growth in serum-free media. Therefore, because direct counts 30 - of cell numbers were not possible, their growth was assayed in 35-mm dishes by using uptake of low specific activity [3H]- thymidine over a 2-day period. Growth media in these instances x included, per ml, 50 ng of 2'-deoxycytidine (Sigma), 50 ng of thymidine (Sigma), and 0.01 ,tCi of [3H]thymidine (60 Ci/ 10 - -x mmol; New England Nuclear) (1 Ci = 3.7 X 1010 becquerels). Higher levels of [3H]thymidine proved to be toxic over extended labeling periods (see also ref. 18). Material insoluble in cold 5% trichloroacetic acid was dissolved in 0.5 ml of 0.1 M NaOH per E 35-mm plate and the radioactivity of 100 ,l was measured in 10 ml of scintillation cocktail (Biofluor; New England Nuclear) 0. in an Intertechnique Instruments (Dover, NJ) model SL30 liquid scintillation spectrophotometer. 0~~~~~~~~~~~~~~~~~00 Cell number and [3H]thymidine incorporation are directly xw23 ,7/ a) proportional in the serum-free media used in this study. 1 / x Comparison of the two growth assays of CHEF/18 cells by using linear regression analysis provided a linear relationship with good fit (regression coefficient = 0.96). Cells grown with 10% serum present incorporated only 70% of the expected ra- dioactivity, perhaps due to dilution of the [3H]thymidine by thymidine in the serum. Flow Microfluorimetric Analysis. Samples to be analyzed for cell-cycle distribution by using flow microfluorimetric analysis were grown in 60-mm dishes as described above and prepared by using a modification of the method described by 1 2 3 4 5 Fried et al. (19), and the DNA distribution frequency (always Time, days within 2 days of sampling) was analyzed by using a model FIG. 1. Growth of Syrian hamster fibroblasts in serum-free 4800A Cytofluorograf (Ortho Instruments, Westwood, MA). media. Each cell line was seeded into 35-mm petri dishes containing alpha/F12 + 10%o calf serum. At "0 hr," after approximately 1 day for RESULTS BHK-21 and 12 hr for PyBHK, the monolayers were washed exten- sively and fed with media as noted below. Cell growth was monitored Growth of Syrian Hamster and Diploid Chinese Hamster at daily intervals. X, BHK-21; 0, PyBHK; -, alpha/F12 + 10% calf Embryo Fibroblasts in Serum-Free Medium. A growth me- serum; - -, alpha/F12 + 4F; - - -, alpha/F12 + transferrin and dium supplemented by FGF, EGF, insulin, and transferrin (4F) FeSO4. Downloaded by guest on September 26, 2021 Cell Biology: Cherington et al. Proc. Natl. Acad. Sci. USA 76 (1979) 3939 T 10- T

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-Transferri n and FeSO4 0 1 2 3 4 5 FIG. 3. Growth factor requirements of Syrian hamster fibroblasts. Time, days Cells were plated in alpha/F12 + 10% calf serum, incubated for 1 day, FIG. 2. Growth of Chinese hamster embryo fibroblasts in and shifted into experimental conditions. Cell numbers (X lo-4) per serum-free media. Procedures were identical to those described for cm2 (+SD) at the time of the shift were: BHK-21, 1.0 i 0.1 (horizontal BHK-21 cells in Fig. 1. X, CHEF/18; 0, CHEF/16; , alpha/F12 + lines); MT1-C2, 0.3 + 0.05 (cross-hatched bars); PyBHK, 1.2 + 0.1 10% fetal calf serum; - -, alpha/F12 + 4F. (hatched bars). Plates in duplicate were counted 44-47 hr after the shift. Error brackets are SD.

Transformation of BHK-21 by Polyoma Virus Results in or without EGF. CHEF/16 exhibited insulin and transferrin- Loss of All but the Transferrin Requirement. The serum-free iron requirements, as did CHEF/18. medium has been used to show that BHK-21 fibroblasts trans- The growth factor requirements of two chemically trans- formed by polyoma virus lose their requirements for most formed diploid subclones of CHEF/18 further supported a growth factors. Fig. 3 compares the growth of three cell lines relationship between transformed phenotype and EGF de- over 2 days in serum-free media with various growth factors deleted. The 4F-supplemented medium yielded about a 4-fold 15r increase in cell number over 2 days for BHK-21, about half that observed with 10% serum (not shown). Some decreased growth T FGF or EGF. Growth of 2 was observed upon deletion of either 0 BHK-21 was also decreased upon deletion of transferrin and +100) FeSO4 and was especially decreased upon deletion of insulin. In contrast, growth of PyBHK was not affected upon deletion of FGF, EGF, or insulin, either singly or in any combination (data not shown). Deletion of the transferrin and iron supple- C) ments significantly decreased the growth of these virally T transformed cells. EGF Requirement Changes Are Most Closely Related to 0 Transformation. A major difference in the EGF requirement x 0- exists between the transformed, tumorigenic, CHEF/16 and the nontumorigenic CHEF/18 clone (Fig. 4). CHEF/18 cell number increased about 4-fold in alpha/F12 + 4F over ap- proximately 2 days, approximately one less doubling than in 10% fetal calf serum (not shown). Deletion of FGF may have somewhat improved growth for CHEF/18; none of the Chinese FIG. 4. Growth factor requirements of Chinese hamster embryo hamster lines required FGF. Deletion of EGF, however, al- fibroblasts. Conditions and procedures were identical to those de- lowed less than one doubling of the population over 2 days. A scribed for Fig. 3 except fetal calf serum replaced calf serum. All plates strong requirement for insulin was also evident. When trans- in duplicate were counted 40-46 hr after shift to experimental con- ditions. CHEF/18 data are expressed as the mean of duplicate ex- ferrin and iron supplements were deleted, there also was sig- periments; CHEF/16 data are expressed as the mean of duplicate nificantly less growth of CHEF/18. CHEF/16, unlike plates; error brackets are SD. Cell numbers (X 10-4) per cm2 (±SD) CHEF/18, did not exhibit a requirement for EGF, as indicated at the time of shift to experimental conditions were: CHEF/18, 2.2 by equivalent cell numbers after 2 days in alpha/F12 + 4F with + 0.1 (stippled bars); CHEF /16, 1.5 ± 0.02 (hatched bars). Downloaded by guest on September 26, 2021 3940 Cell Biology: Cherington et al. Proc. Natl. Acad. Sci. USA 76 (1979) T 8-

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xI.CV 4- E I jr - c 2 1 2 DNA content E FIG. 6. Cell-cycle distribution of cells placed into media with -= 2- various growth factors deleted. Exponentially growing cells were It plated into 60-mm dishes in alpha/F12 + 10%o serum. The dishes were washed after approximately 1 day and cells were fed with either alpha/F12 + 4F (-) or alpha/F12 + transferrin and FeSO4 (--). -r (A) BHK-21, (B) CHEF/18, (C) CHEF/16, (D) T30-4. Samples were prepared for flow microfluorimetric analysis after 1 day in experi- mental conditions. -tur ana insulin FIG. 5. Growth factor requirements of chemically transformed upon serum deprivation (4, 20-24), this is not true for cells CHEF/18. Conditions for [3H]thymidine incorporation were described transformed by other means (4-7, 25). EGF and insulin will in Materials and Methods. Growth conditions were as described in permit Swiss 3T3 mouse cells to continue to grow under Fig. 4. All plates were sampled in duplicate 42-52 hr after shift to serum-deprived conditions that normally arrest these cells in experimental conditions. Data for CHEF/18 are expressed as the Go (P. W. Rossow and A. B. Pardee, unpublished data). BHK-21 means of separate experiments. Data for other cell lines are expressed and CHEF/18 fibroblasts also require EGF and insulin for as the means of duplicate plates i SD (brackets). Horizontal lines, CHEF/18; cross-hatched bars, 21-2; stippled bars, 21-2-M3; hatched growth (Figs. 3 and 4); when these factors were deleted, cells bars, T30-4. were arrested in Go (Fig. 6 A and B). CHEF/16 cells that lacked a requirement for EGF were arrested in Go when deprived of FGF, EGF, and insulin (Fig. 6C) or if only insulin was removed. pendence (Fig. 5). Clones 21-2 and 21-2-M3 were selected after Retention of growth arrest in Go was also shown by the ethyl methanesulfonate mutagenesis; they form tumors in nude tumor-derived clone T30-4 (Fig. 6D). This clone, like mice. Clones 21-2 and 21-2-M3 retained the requirements for CHEF/16, expressed no requirement for FGF or EGF (Fig. EGF and insulin expressed by the parent line (CHEF/18). 5) and thus the Go arrest shown in Fig. 6 was a result of insulin However, the EGF requirement appeared to be less stringent deprivation. for the chemically transformed lines. Growth of 21-2-M3 in a nude mouse yielded the rapidly growing clone T30-4. This DISCUSSION clone lost the EGF requirement expressed by the parent line Transformation of cells by DNA tumor viruses reduces the but had retained the insulin requirement. This requirement for requirement for serum and abolishes GI-associated growth insulin is also indicated by the G1 arrest of these cells upon regulatory events. The basis for decreased serum requirement growth factor deprivation (Fig. 6). All four lines expressed a of polyoma-transformed BHK-21 is that these cells no longer requirement for transferrin. Requirements for factors supplied required the FGF, EGF, and insulin that were required by by serum and not by 4F were more stringent in the chemically BHK-21. They retained a requirement for transferrin. Chem- transformed cells (data not shown). These requirements are not ical or "spontaneous" transformation of Chinese hamster fi- correlated with reduced serum requirement or tumorigenicity, broblasts mainly decreased the EGF requirement. These lines however, and are possible secondary effects of mutagenesis. retained an insulin requirement as well as a transferrin re- These results suggest that a continuum of phenotypes exists quirement. Thus transformation by the DNA tumor virus with respect to growth factor requirements and transformation, (polyoma) was not typical of transformation by other means. a continuum leading from nontumorigenic andc high EGF re- Transferrin was required by transformed and untransformed quirement to tumor-derived and no EGF requirement. This cells alike. This growth factor acts differently from FGF, EGF, continuum is also indicated with MT1-C2, a clone derived from and insulin in that (i) progress of cells through G2 is inhibited a tumor formed in a nude mouse by BHK-21. This clone re- in the absence of transferrin and adequate iron (ref. 26; un- tained the insulin and transferrin requirements expressed by published results) and (ii) PyBHK cells, which are not able to the parent BHK-21 line (Fig. 3). The FGF and EGF require- accumulate in G1 upon serum, nutrient, or growth factor star- ments were also evident, although they appeared to be de- vation, retained only their requirement for transferrin. This is creased. not particularly surprising because transferrin is involved with Transformed CHEF Cell Lines Retain the Ability to Arrest delivery of essential iron to the cells (26, 27). Growth in GI. Whereas cells transformed by DNA tumor vi- The diminished requirement for EGF appeared most closely ruses appear to have lost the ability to arrest growth in G1 (Go) related to the degree of transformation. This was particularly Downloaded by guest on September 26, 2021 Cell Biology: Cherington et al. Proc. Nati. Acad. Sci. USA 76 (1979) 3941

evident upon comparison of the extremes-i.e., the tumorigenitc 6. IOernandez-Pol, J. A., Bono, V. H. & Johnson, G. S. (1977) Proc. CHEF/16 cells or the tumor-derived T30-4 cells vs. the Natl. Acad. Sci. USA 74,2889-2893. CHEF/18 cells. A continuum of changes in 7. Moses, H. L., Proper, J. A., Volkenant, M. E., Wells, D. J. & Getz, nontumorigenic M. J. (1978) Cancer Res. 38, 2807-2812. the EGF requirement is indicated for the chemically trans- 8. Scher, C. D., Pledger, W. J., Martin, P., Antoniades, H. & Stiles, formed Chinese hamster cells (21-2 and 21-2-M3) and for the C. D. (1978) J. Cell. Physiol. 97, 371-380. BHK-21 derivative line (MT1-C2) cloned from a tumor grown 9. Iudland, P. S., Eckhart, W., Gospodarowicz, D. & Seifert, W. in a nude mouse. Tumorigenicity apparently can result from (1974) Nature (London) 250,26-31. only a partial loss of a requirement for a growth factor such as 10. Todaro, G. J., DeLarco, J. E. & Cohen, S. (1976) Nature (London) EGF. A reduction in the requirement might permit excessive 264,26-31. growth of these cells in vivo under conditions that do not permit 11. Stiles, C. D., Capone, G. T., Scher, C. D., Antoniades, H. N., Van Wyk, J. J. & Pledger, W. J. (1979) Proc. Nati. Acad. Sci. USA 76, growth of growth factor-dependent cells. 1279-1283. Various cell lines require EGF and insulin to transit the G1 12. Ames, B. N. (1979) Science 204,587-593. phase of the cell cycle (this work; P. W. Rossow and A. B. Par- 13. Oppermann, H., Levinson, A. D., Varmus, H. E., Levintow, L. dee, unpublished data). However, the GI-associated require- & Bishop, J. M. (1979) Proc. Natl. Acad. Sci. USA 76, 1804- ments for these factors are separable, because the requirement 1808. for EGF may be abolished while the insulin requirement is 14. Hayashi, I. & Sato, G. H. (1976) Nature (London) 259, 132- retained. Certain transformed cells retain the ability to arrest 134. growth at a restriction point in G1 (4, 20, 28). This may be ex- 15. Stoker, M. G. D. & Macpherson, I. A. (1964) Nature (London) 203, 1355-1357. plained by the fact that some transformed cells retain an insulin 16. Sager, R. & Kovac, P. E. (1978) Somatic Cell Genet. 4, 375- requirement for transit through G1 even if their EGF re- 392. quirement is lost. Other transformants, however (e.g., PyBHK), 17. Schneider, E. L., Stanbridge, E. J. & Epstein, C. J. (1974) Exp. have lost the requirements for all growth factors associated with Cell Res. 84,311-318. GI transit and stimulation of DNA synthesis (29), and thus have 18. Pollack, A., Bagwell, C. B. & Irvin, G. L., III (1979) Science 203, lost their G1 growth regulation. 1025-1027. Cell hybridization techniques can be used to study segrega- 19. Fried, J., Perez, A. G. & Clarkson, B. D. (1976) J. Cell Biol. 71, 172-181. tion of a variety of properties associated with transformation 20. Pardee, A. B. (1974) Proc. Natl. Acad. Sci. USA 71, 1286- (16, 30, 31), including tumorigenicity and diminished re- 1290. quirements for serum and specific growth factors such as EGF. 21. Pardee, A. B. & James, L. J. (1975) Proc. Natl. Acad. Sci. USA In a preliminary report, the loss of the EGF requirement in 72,4994-4998. progeny form somatic cell hybrids between normal and tu- 22. Bartholomew, J. C., Yokota, H. & Ross, P. (1976) J. Cell. Physiol. morigenic cells has been described (31). 88,277-286. 23. Martin, R. G. & Stein, S. (1976) Proc. Natl. Acad. Sci. USA 73, The authors thank David S. Schneider for help in preparation of the 1655-1659. manuscript. This work was supported by U.S. Public Health Service 24. Bush, H. & Shodell, M. (1977) J. Cell. Physiol. 90,573-584. Grant GM 24571 to A.B.P., National Research Service Award Pre- 25. Mierzejewski, K. & Rozengurt, E. (1976) Biochem. Biophys. Res. doctoral Training Grant S T32 GM 07306-04 to P.V.C., and Medical Commun. 73, 271-278. Scientist Training Program Predoctoral Training Grant 5 T05 GM 26. Rudland, P. S., Durbin, H., Clingan, D. & de Asua, L. J. (1977) 02220-05 to B.L.S. Biochem. Biophys. Res. Commun. 75,556-562. 27. Messmer, T. 0. (1973) Exp. Cell Res. 77, 404-408. 1. Clarke, G. D., Stoker, M. G. P., Ludlow, A. & Thornton, M. (1970) 28. Pardee, A. B., Dubrow, R., Hamlin, J. L. & Kletzien, R. F. (1978) Nature (London) 227,798-801. Annu. Rev. Biochem. 47,715-750. 2. Dulbecco, R. (1970) Nature (London) 227, 802-806. 29. Gospodarowicz, D. & Moran, J. S. (1976) Annu. Rev. Biochem. 3. Green, M. (1970) Annu. Rev. Biochem. 39, 701-756. 45, 531-558. 4. Dubrow, R., Riddle, V. G. H. & Pardee, A. B. (1979) Cancer Res. 30. Howell, A. N. & Sager, R. (1979) Somatic Cell Genet. 5, 129- 39,2718-2726. 143. 5. Holley, R. W., Baldwin, J. H., Kiernan, J. A. & Messmer, T. 0. 31. Sager, R., Cherington, P. V., Smith, B. L. & Pardee, A. B. (1979) (1976) Proc. Natl. Acad. Sci. USA 73,3229-3232. Fed. Proc. Fed. Am. Soc. Exp. Biol. 38, 635 (abstr.). Downloaded by guest on September 26, 2021