Suppression of the expression of erythroid functions in murine ...

Proc. Natl. Acad. Sci. USA Vol. 76, No. 4, pp. 1906-1910, April 1979

Cell Biology

Tumor promoter-mediated inhibition of cell differentiation: Suppression of the expression of erythroid functions in murine erythroleukemia cells (Friend virus/globin mRNA/spectrin/heme/hexamethylene bisacetamide)

EITAN FIBACH*t, ROBERTO GAMBARI*, PHYLLIS A. SHAW*, GEORGE MANIATIS**, ROBERTA C. REUBEN*, SHIGERU SASSA§, RICHARD A. RIFKIND*, AND PAUL A. MARKS* *Departments of Medicine and of Human Genetics and Development and the Cancer Center, Columbia University, New York, New York 10032; and §The Rockefeller University, New York, New York 10021 Contributed by Paul A. Marks, January 30, 1979

ABSTRACT Previous studies demonstrated that 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumor promoter, is a potent inhibitor of inducer-mediated differentiation of murine erythroleukemia cells. Inhibition of cell differentiation was associated with inhibition of cell growth. The present studies, employing a cell line adapted for growth in TPA, demonstrate that inhibition of differentiation is not dependent upon inhibition of cell growth or a change in the cellfdivision cycle; neither is inhibition of differentiation accompanied by detectable effect on cell uptake of [3H]hexamethylene bisacetamide, the inducer used in these studies. TPA causes an inhibition of expression of all hexamethylene bisacetamide-inducible erythroid characteristics measured, including commitment to terminal cell division, accumulation of globin mRNA, and synthesis of globins, spectrin, heme synthetic enzymes (5-aminolevulinic acid dehydratase and uroporphyrinogen-I synthase) and heme. A hypothetical model for the inhibitory action of tumor promoters on terminal cell differentiation is discussed.

MATERIALS AND METHODS MELC, subclone DS19-1OTS, a TPA-sensitive cell line isolated from strain 745A-DS19, was maintained in culture and characterized as described (27). DS19-1OTS(TPA) cells were obtained by passaging DS19-IOTS cells for at least 1 month in medium containing TPA at 100 ng/ml. Cells were grown in suspension culture by diluting the cells twice a week at 105 cells/ml in fresh medium. Cultures for experiments summarized below were inoculated from 1 day, logarithmic phase, cultures at 105 cells/ml. HMBA was prepared as described (13). TPA was obtained from the Consolidated Midland Co. (Brewster, NY). TPA was dissolved in dimethyl sulfoxide to a concentration of 1 mg/ml and further diluted in acetone to 100 ,ug/ml. Final concentrations of dimethyl sulfoxide and acetone in culture medium were 0.01% and 0.1%, respectively. These concentrations of either solvent had no detectable effect on cell growth or accumulation of benzidine-reactive cells. In these studies, 4 mM HMBA and TPA at 100 ng/ml were used unless otherwise noted. Benzidine-reactive cells were assayed as described (27). Commitment to differentiate, defined as the ability to express the program of erythroid differentiation after removal of the inducing agent, was assayed by the method of Fibach et al. (25). Distribution of cells with respect to the cell cycle was assayed by determination of DNA content per cell by employing pro-

12-0-Tetradecanoyl-phorbol-13-acetate (TPA) is one of a group of plant diterpenes that are tumor promoters in the two-stage mouse skin carcinogenesis system (1-4). TPA is a reversible inhibitor of cell differentiation in several in vitro systems, including murine erythroleukemia cells (MELC) (5, 6), murine neuroblastoma cells (7), chicken myoblasts and chondroblasts (8, 9), and the fibroblast-adipocyte conversion (10). It has been postulated that tumor promotion itself may involve interference in the process of differentiation (1, 6, 11). Of the several systems examined to date, MELC is the best characterized with respect to the molecular events during expression of differentiation. Upon exposure to various agents such as dimethyl sulfoxide (12), hexamethylene bisacetamide (HMBA) (13), or butyric acid (14), these cells initiate erythroid differentiation, including characteristic morphological changes (12), synthesis and accumulation of globin mRNA (15-18), synthesis of a and ( globins (19, 20), increase in spectrin content (21, 22), increase in the activity of heme synthetic enzymes and heme content (23, 24), and loss of the capacity for cell division (12, 25, 26). We have previously found that TPA-mediated inhibition of differentiation is accompanied by an early and pronounced inhibition of cell growth (6, 27). In the present studies we demonstrate that an effect on cell growth is not required for the effect on differentiation. Furthermore, we show that, under conditions in which TPA has no detectable effect on cell growth or uptake of inducer, it inhibits the expression of many characteristics of erythroid differentiation, including commitment to terminal cell division and accumulation of globin mRNA, globin, spectrin, heme synthetic enzymes, and heme.

pidium iodide and flow microfluorometry (28, 29). Accumulation of newly synthesized cytoplasmic total RNA and globin mRNA were measured by employing oligo(dT)cDNA-cellulose chromatography (30). Pulse-chase studies were performed as follows: Cells were cultured with HMBA plus TPA for 48 hr, recovered, and resuspended in [3H]uridinecontaining medium for 15 min to label newly synthesized RNA. The cells were again recovered, resuspended in medium containing 20 mM (nonradioactive) uridine and HMBA without or with TPA, and then incubated for 6 hr. Globin synthesis was measured by described methods (16, 31). Pulse-chase experiments for study of the stability of newly synthesized globin were performed similarly to those for newly synthesized globin mRNA, except that [35SImethionine was employed as a source of radioactivity, labeling was for 30 min, and the period of chase was 5 hr. Assays for the activities of 3-aminolevulinic acid Abbreviations: MELC, murine erythroleukemia cells; HMBA, hexamethylene bisacetamide; TPA, 12-0-tetradecanoyl-phorbol-13-acetate; ALA-D, 6-aminolevulinic acid dehydratase; Uro-S, uroporphyrinogen-I synthase. t Present address: Hadassah University Hospital, Mt. Scopus, Jerusalem, Israel. t Present address: University of Patras Medical School, Patras,

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Greece.

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Cell Biology: Fibach et al. dehydratase (ALA-D) and uroporphyrinogen-I synthase (Uro-S) and for heme concentration were carried out in duplicate (24). The distribution of the cells with respect to spectrin content was determined by an indirect immunofluorescence method (22). These cells were treated with antispectrin antibodies followed by staining with a fluorescein-conjugated sheep IgG. The intensity of cellular fluorescence was measured by flow microfluorometry. Cell uptake of HMBA labeled with 3H in the terminal methyl group of acetate was measured by described techniques (32). [3H]HMBA was added to a final concentration of 5 mM at 1 MiCi/ml (1 Ci = 3.7 X 1010 becquerels). RESULTS Cell Growth. MELC cells (strain DS19-10TS) cultured with TPA with or without HMBA exhibited a lag in onset of logphase growth of up to 3 days (Fig. 1A). Cells cultured without addition of inducer or tumor promoter exhibited little lag in onset of cell growth; cells cultured with HMBA alone, as reported (28), exhibited a lag in onset of log-phase growth of approximately 24 hr. To examine whether TPA-mediated inhibition of differentiation was related to the effect of TPA on cell growth, we performed the following experiments. DSL9-1OTS cells were grown with TPA (100 ng/ml) for at least 30 days. Cells adapted to growth in the presence of TPA in this manner, designated DS19-LOTS(TPA), exhibited similar growth curves whether cultured without or with TPA (Fig. 1B). DS19-LOTS(TPA) cells, cultured with HMBA, either without or with TPA, also exhibited similar growth curves. The transient inhibition of onset of log-phase growth of DS19-IOTS cells caused by TPA is due to a delay in entry of the TPA-treated cells into S phase. This is shown by analysis of the distribution of cells with respect to the cell division cycle at time intervals after onset of culture with HMBA, without and with TPA. DS19-1OTS cells cultured with HMBA plus TPA accumulate in GI phase, detectable at 24 hr and persisting until about 40 hr (data not shown). By 48 hr in culture, these cells are proceeding through S phase with kinetics similar to those of cells cultured with HMBA alone. By comparison, DS19-10TS(TPA) cultured with HMBA proceed through the cell division cycle with similar kinetics whether in the presence or absence of TPA. DS19-1OTS(TPA) cells cultured with HMBA, without or with

Proc. Natl. Acad. Sci. USA 76 (1979)

1907

ATP, exhibited the characteristic HMBA-mediated lag in onset of log-phase growth (Fig. 1B). This lag has been shown to be due to an inducer-mediated transient prolongation of G1 (28). Benzidine-Reactive Cells. Benzidine-reactive DS191OTS(TPA) cells began to accumulate between 24 and 48 hr of culture with HMBA; the proportion of reactive cells exceeded 90% by 5 days. The accumulation of benzidine-reactive DS19-lOTS(TPA) cells in cultures with HMBA was markedly inhibited by TPA (Fig. 2). As reported (6, 27), TPA-mediated inhibition of HMBA-induced differentiation of DS1910TS(TPA) cells was reversible. Cells cultured with HMBA and TPA for as long as 21 days displayed fewer than 10% benzidine-reactive cells. When these cells were transferred to fresh medium with HMBA alone, they were induced to become benzidine reactive at a rate similar to that observed in cells not previously cultured with TPA. Cells have been grown with HMBA and TPA for periods of 3 months or longer, and TPAmediated inhibition of differentiation was reversed when cells were transferred to medium containing HMBA without tumor promoter. Commitment to Erythroid Differentiation. DS1910TS(TPA) cells were cultured with HMBA, and aliquots from these cultures were removed after different periods of time to assay for the proportion of cells committed to terminal cell division and hemoglobin synthesis. Cultures with HMBA exhibited an increase in committed cells as early as 16 hr; by 60 hr, 98% of the cells were committed (Fig. 3A). The kinetics of HMBA-mediated commitment of DS19-1OTS(TPA) cells are similar to those reported for the parental MELC DS19 strain (25). To examine the relationship between TPA-mediated inhibition of HMBA-induced differentiation and commitment, DSl9-10TS(TPA) cells were cultured with HMBA, and, at intervals up to 72 hr, aliquots of the cells were transferred to semisolid cloning media without or with TPA at 100 ng/ml and without inducer (Fig. 3A). After a 46-hr exposure to HMBA, 55% of the cells displayed commitment by cloning into medium free of HMBA or TPA. If cells were cloned into TPA-containing medium (HMBA-free), less than 10% of the cells would express that commitment. After 60 hr in HMBA, 98% of the cells were committed, but only 48% of the cells would show evidence of that commitment if they were cloned in medium containing TPA. By 72 hr of exposure to HMBA, transfer to TPA-containing semisolid medium could not suppress the expression of

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Time, days FIG. 1. Effect of TPA on growth of DS19-lOTS (A) and DSl91OTS(TPA) (B) cells. Cells were cultured in medium without additions (3), with 5 mM HMBA (0), with TPA at 100 ng/ml (-), and with 5 mM HMBA plus TPA at 100 ng/ml (0).

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FIG. 2. Effect of TPA on HMBA induction of benzidine-reactive cells. DS19-10TS(TPA) cells were cultured with 4 mM HMBA (0) or with 4 mM HMBA plus TPA (100 ng/ml) (-). At times indicated by the arrows (3, 5, and 21 days), cells were transferred from medium containing HMBA plus TPA to fresh medium with 4 mM HMBA.

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Cell'Biology:

Fibach et al.

Proc. Natl. Acad. Sci. USA 76 (1979)
85%). In a parallel series of experiments, cells were cultured in suspension with both HMBA and TPA, and, at intervals up to 72 hr, aliquots were transferred to semisolid cloning medium without or with TPA and without inducer (Fig. 3B). Cells transferred after 35, 46, 60, 62, and 72 hr to medium without TPA showed progressively increasing proportions of differentiated colonies. Cells transferred into medium with TPA showed little increase in differentiated colonies; less than 10% by 60-72 hr. Globin mRNA. DS19-10TS(TPA) cells were cultured with HMBA without or with TPA for 24, 48, 72, and 96 hr. At each time, aliquots of the cells were recovered and incubated for 2 hr with [3H]uridine to label newly synthesized RNA. The rate of accumulation of newly synthesized globin mRNA was lower in cells cultured with inducer and tumor promoter compared with cells cultured with inducer alone (Fig. 4A). To determine whether the inhibitory effect of TPA on accumulation of globin mRNA was due to a decrease in stability of newly synthesized globin mRNA, we performed the following experiments. DS19-1OTS(TPA) cells were cultured with HMBA without or with TPA for 48 hr. The cells were "pulsed" with [3H]uridine for 15 min to label newly synthesized RNA and then "chased" by culture with 20 mM (nonradioactive) uridine for 6 hr. The levels of newly synthesized (radioactive) globin mRNA and total RNA were unchanged throughout this 6-hr period (data not shown), in agreement with the findings of Lowenhaupt and Lingrel (33); TPA did not alter this stability of globin mRNA. These findings indicate that TPA-mediated inhibition of newly synthesized globin mRNA does not reflect a decrease in the stability of globin mRNA. It appears likely that TPA acts to inhibit globin mRNA accumulation at the level of transcription or nuclear processing. Globin. The rate of accumulation of newly synthesized globin and the globin content of DS19-1OTS(TPA) cells cultured with HMBA and TPA were lower than that of cells cultured with HMBA alone (Fig. 4B). The rates of synthesis of each of the globin chains, a, fmaj and Pmin, were inhibited by TPA (data not shown). TPA had no detectable effect on the stability of newly synthesized globin. This was indicated by the following experiments. DS19-10TS(TPA) cells were cultured with HMBA

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FIG. 4. Effect of TPA on HMBA-induced accumulation of newly synthesized globin mRNA (A) and globin (B). DS19-1OTS(TPA) cells were cultured with 4 mM HMBA without (0) or with TPA (100 ng/ml) (e). The level of globin mRNA is expressed as the percentage of that radioactivity in cytoplasmic RNA recovered in globin mRNA. Globin content is expressed as the percentage of [35S]methionine incorporated into total protein that is present in globin.

without or with TPA; at 48 hr, the cells were "pulsed" with [15S]methionine for 5 hr. The level of radioactive globin remained unchanged during the 5-hr period of "chase," with or without TPA. Heme Synthetic Enzymes and Heme. The induction of MELC to differentiate is accompanied by an increase in activity of enzymes of the heme synthetic pathway and in the accumulation of heme. DS19-10S(TPA) cells cultured with HMBA and TPA have lower levels of activity of two of these enzymes, ALA-D and Uro-S, and a lower heme content than cells cultured with HMBA alone (Fig. 5). Inhibition of accumulation of heme is most marked at 60 hr, at which time the heme content of cells cultured with inducer and TPA is 40% of that in cells cultured with inducer alone. TPA-mediated inhibition of the increase of ALA-D and Uro-S activities was observed at or prior to 60 hr, but inhibition was minimal at 90 hr. Spectrin. The spectrin content of DS19-10TS(TPA) cells cultured with HMBA without and with TPA was determined at intervals up to 96 hr. In preliminary experiments it was shown that TPA has no direct effect on the reaction between spectrin and antispectrin antibodies (data not shown). Cells exposed to HMBA and TPA display a lower level of spectrin-specific immunofluorescence, compared to cells cultured with HMBA alone (Fig. 6). The unimodal distribution of cells with respect to spectrin immunofluorescence is not altered by TPA, but the curve is shifted to a lower modal value. Cell Uptake of HMBA. To determine whether the inhibitory effect of TPA on HMBA-mediated differentiation is due to an action of the tumor promoter on the uptake of inducer by MELC, we performed the following experiments. DS191OTS(TPA) cells were cultured with [3H]HMBA, without or with TPA, and aliquots were removed to determine the amount of radioactivity taken up in the cell. The curve of increase in radioactivity of the cells was similar for cells cultured with or without the tumor promoter. A plateau level of radioactivity was achieved by 24 hr in cultures with and without TPA. TPA does not alter the kinetics of uptake of HMBA under the conditions of these experiments.

Cell Biology: Fibach et al.

Proc. Nati. Acad. Sci. USA 76(1979)

different chromosomes, responsible for various characteristic erythroid cell properties (34). TPA mediates suppression of the expression of all of those characteristics measured, including terminal cell division, synthesis of globin mRNA and globins,

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DISCUSSION Previous studies from our laboratory (6, 27) and from other groups (5, 7-10) have demonstrated that TPA is a potent inhibitor of terminal differentiation of MELC and various other cell culture systems. The present studies show that this inhibitory effect is specific and not a consequence of a toxic effect on cell growth. MELC can be adapted to growth in TPA; such adapted cells remain sensitive to HMBA-mediated induction of differentiation in the absence of TPA. Addition of TPA to these adapted cells in culture with HMBA causes inhibition of differentiation without detectable effect on cell growth or cell division cycle. HMBA-mediated differentiation of MELC involves the coordinated expression of a number of genes, some, at least, on

increased activities of heme synthetic enzymes (ALA-D and Uro-S), and the accumulation of heme and of benzidine-reactive (hemoglobin-containing) cells. These characteristics of differentiation are not uniformly suppressed by TPA. The accumulation of benzidine-reactive cells is most sensitive, whereas the heme synthetic enzymes, globin mRNA accumulation, and globin synthesis appear relatively less suppressed. Differences in the apparent degree of suppression of the several characteristics assayed may reflect differences in the sensitivities of the several assays or differences in the stringency of control exercised by the TPA-sensitive reaction on the expression of different erythroid characteristics. Evidence from a number of studies employing variant MELC lines and different inducers has demonstrated that uncoordinated expression of these characteristics of differentiation, such as terminal cell division, globin mRNA, and spectrin, can occur (35-37), indicating that the relative extent of expression of these characteristics can vary. Studies employing the assay for commitment in the presence and absence of TPA suggest that the tumor promoter has an effect on already committed cells, causing either a reversal of commitment or a suppression of the expression of characteristics of the committed state, specifically (in the present assay) terminal cell division and hemoglobin production. Another inhibitor of induced MELC differentiation, hydrocortisone, appears to share this property (38). The studies on transfer of MELC cells from medium containing HMBA or HMBA and TPA into medium without these agents strongly suggest that MELC cells retain, for a period, a "memory" of past exposure to HMBA. The "memory" for prior exposure to HMBA is indicated by the finding that cells cultured with HMBA and TPA do not differentiate, but, when such cells are then transferred to medium without these agents, a portion differentiates (see Fig. 3B). The evidence is equivocal with respect to a "memory" for prior exposure to TPA in that cells transferred from suspension with HMBA and TPA into suspension with HMBA alone show no suppression of differentiation as result of prior culture with TPA (see Fig. 2). Under cloning conditions, however, cells transferred from HMBA and TPA to fresh semisolid medium without either agent display a lower proportion of differentiated colonies than cells transferred from HMBA alone. This may reflect a partial memory for prior exposure to TPA under cloning conditions, or, perhaps, provides evidence for

instability of the HMBA-effect, which becomes reversible if it is not expressed promptly enough. A model for the interaction of MELC cells with inducers and TPA-like inhibitors can be proposed, which, though speculative, is based upon the known effects of these agents and serves to focus future experimental approaches. Certain inducers, at least, such as the polar-planar compounds (e.g., HMBA), may interact with the cell at the level of the plasma membrane. The evidence for this is largely circumstantial (reviewed in ref. 34) and includes early effects on membrane transport (39), lectin-mediated reactions (40), inducer activity of established membrane-target agents such as ouabain (41), and cyclic nucleotide content (42). It is postulated that the generation of a transmembrane signal (? cyclic AMP) is responsible for an alteration in a nuclear, chromatin-level, function. The product of this function represents the molecular basis for "commit-

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Cell Biology: Fibach et al.

of erythroid cells. [Some inducers of MELC cell differentiation such as actinomycin D (43) may act directly at the nuclear level.] TPA, whether it has a primary cellular target at the plasma membrane, as has been postulated (11), or at some other yet to be determined site, may act to alter the product responsible for initiating the "expression" of commitment and, thus, block differentiation. A brief delay in the onset of log-phase growth, due to a transient prolongation in the G1 phase of the cell cycle, is an early response to HMBA and other inducers (28, 42, 45). This cell cycle effect of inducer is not suppressed by TPA. Whether this indicates that the cell cycle effect of inducers is an intrinsic component of the commitment phase of differentiation induction and is not susceptible to TPA-mediated suppression (according to the above hypothetical model) or, alternatively, indicates that the G1 delay is a nonspecific and unrelated effect of inducers, remains to be determined. These studies were supported, in part, by Grants CA-13696 and CA-18314 from The National Cancer Institute, Grant CH-68 from The American Cancer Society, and Grant PCM-75-08696 from The National Science Foundation. E.F. was a Schultz Foundation Fellow. R.G. is a Visiting Fellow from the Istituto di Biologia Generale, Policlinico, Umberto I, Rome, Italy. R.C.R. is a Special Fellow of the Leukemia Society of America, Inc. 1. Berenblum, I. (1969) Prog. Exp. Tumor Res. 11, 21-30. 2. Boutwell, R. K. (1974) CRC Crit. Rev. Toxicol. 2,419-443. 3. Hecker, E. (1975) in Handbuch der Allegemeinen Pathologie, ed. Brundmann, E. (Springer, Berlin, West Germany), Vol. IV/ 16, pp. 651-676. 4. Van Duuren, B. L. (1969) Prog. Exp. Tumor Res. 11, 31-38. 5. Rovera, G., O'Brien, T. A. & Diamond, L. (1977) Proc. Natl. Acad. Sci. USA 74,2894-2898. 6. Yamasaki, H., Fibach, E., Nudel, U., Weinstein, I. B., Rifkind, R. A. & Marks, P. A. (1977) Proc. Natl. Acad. Sci. USA 74, 3451-3455. 7. Ishii, D., Fibach, E., Yamasaki, H. & Weinstein, I. B. (1978) Science 200,556-559. 8. Cohen, R., Pacifici, M., Rubenstein, N., Biehl, J. & Holtzer, H. (1977) Nature (London) 266,538-540. 9. Pacifici, M. & Holtzer, H. (1977) Am. J. Anat. 150,207-212. 10. Diamond, L., O'Brien, T. G. & Rovera, G. (1977) Nature (London) 269, 247-248. 11. Weinstein, I. B., Wigler, M., Fisher, P., Sisskin, E. & Pietropaolo, C. (1978) in Carcinogenesis, eds. Slaga, T. J., Sivak, A. & Boutwell, R. K. (Raven, New York), Vol. 2, pp. 313-333. 12. Friend, C., Scher, W., Holland, J. G. & Sato, T. (1971) Proc. Natl. Acad. Sci. USA 68,378-382. 13. Reuben, R. C., Wife, R. L., Breslow, R., Rifkind, R. A. & Marks, P. A. (1976) Proc. Natl. Acad. Sci. USA 74,862-866. 14. Leder, A. & Leder, P. (1975) Cell 5,319-322. 15. Gilmore, R. S., Harrison, P. R., Eindass, J. W., Affara, M. & Paul, J. (1974) Cell Differ. 3, 23-30. 16. Nudel, U., Salmon, J., Fibach, E., Terada, M., Rifkind, R. A. & Marks, P. A. (1977) Cell 12, 463-469. 17. Ostertag, W., Melderis, H., Steinheider, G., Kluge, N. & Dube, S. (1972) Nature (London) New Biol. 239,231-234. 18. Ross, J., Ikawa, Y. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA

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19. Boyer, S. H., Wuu, K. D., Noyes, A. N., Young, R., Scher, W., Friend, C., Preisler, H. D. & Bank, A. (1972) Blood 40, 832835.

Proc. Natl. Acad. Sci. USA 76 (1979) 20. Kabat, D., Sheraton, C. C., Evans, L. H., Bigley, R. & Koler, R. D. (1975) Cell 5,331-338. 21. Eisen, H., Bach, R. & Emery, R. (1977) Proc. Nat!. Acad. Sci. USA 74,3898-3902. 22. Rifkind, R. A., Fibach, E., Maniatis, G., Gambari, R. & Marks, P. A. (1979) in Conference on Cellular and Molecular Regulation of Hemoglobin Switching, eds. Nienhuis, A. W. & Stamatoyannopoulas, G. (Grune & Stratton, New York), pp. 421436. 23. Ebert, P. S. & Ikawa, Y. (1974) Proc. Soc. Exp. Biol. Med. 146, 601-604. 24. Sassa, S. (1976) Exp. Med. 143,305-315. 25. Fibach, E., Reuben, R., Rifkind, R. A. & Marks, P. A. (1977) Cancer Res. 37, 440-444. 26. Gusella, J., Geller, R., Clarke, B., Weeks, V. & Housman, D. (1976) Cell 9, 221-229. 27. Fibach, E., Yamasaki, H., Weinstein, I. B., Marks, P. A. & Rifkind, R. A. (1978) Cancer Res. 38,3685-3688. 28. Terada, M., Fried, J., Nudel, U., Rifkind, R. A. & Marks, P. A. (1977) Proc. Natl. Acad. Sci. USA 74,248-252. 29. Gazitt, Y., Deitch, A. D., Marks, P. A. & Rifkind, R. A. (1978) Exp. Cell Res. 117,413-420. 30. Gambari, R., Terada, M., Bank, A., Rifkind, R. A. & Marks, P. A. (1978) Proc. Natl. Acad. Sci. USA 75,3801-3804. 31. Nudel, U., Salmon, J., Terada, M., Bank, A., Rifkind, R. A. & Marks, P. A. (1977) Proc. Natl. Acad. Sci. USA 74, 11001104. 32. Reuben, R. C., Marks, P. A., Rifkind, R. A., Terada, M., Fibach, E., Nudel, U., Gazitt, Y. & Breslow, R. (1977) Oji International Seminar on Genetic Aspects of Friend Cells, ed. Ikawa, Y. (Academic, New York), in press. 33. Lowenhaupt, K. & Lingrel, J. B. (1978) Cell 14,337-544. 34. Marks, P. A. & Rifkind, R. A. (1978) Annu. Rev. Biochem. 47, 419-448. 35. Harrison, P. R., Rutherford, T., Conkie, D., Affara, N., Somerville, J., Hissey, P. & Paul, J. (1978) Cell 14, 61-70. 36. Eisen, H., Keppel-Ballivet, F., Georgopoulos, C. P., Sassa, S., Gravich, J., Pragnell, I. & Ostertag, W. (1978) in Cold Spring Harbor Symposia on Cell Proliferation, eds. Clarkson, B., Marks, P. A. & Till, J. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. 5, pp. 277-294. 37. Marks, P. A., Rifkind, R. A., Bank, A., Teroda, M., Gambari, R., Fibach, E., Maniatis, G. & Reuben, R. (1979) in Conference on Cellular and Molecular Regulation of Hemoglobin Switching, eds. Nienhuis, A. W. & Stamatoyannopoulas, G. (Grune & Stratton, New York), pp. 437-456. 38. Santoro, M. G., Benedetto, A. & Jaffe, B. M. (1978) Biochem. Biophys. Res. Commun. 85, 1510-1517. 39. Loritz, F., Bernstein, A. & Miller, R. C. (1977) J. Cell Physiol. 90,423-438. 40. Eisen, H., Nasi, S., Georgopoulos, C. P., Arndt-Jovin, D. & Ostertag, W. (1977) Cell 10, 689-695. 41. Bernstein, A., Hunt, D. M., Crichley, V. & Mak, T. W. (1976) Cell 9,375-381. 42. Gazitt, Y., Reuben, R. C., Deitch, A. D., Marks, P. A. & Rifkind, R. A. (1978) Cancer Res. 38,3779-3783. 43. Terada, M., Epner, E., Nudel, U., Salmon, J., Fibach, E., Rifkind, R. A. & Marks, P. A. (1978) Proc. Natl. Acad. Sci. USA 75, 2795-2799. 44. Houseman, D., Gusella, J., Geller, R., Levenson, R. & Weil, S. (1978) in Cold Spring Harbor Symposia on Cell Proliferation, eds. Clarkson, B., Marks, P. A. & Till, J. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Vol. 5, pp. 193-207. 45. Friedman, E. A. & Schildkraut, C. L. (1978) Proc. Natl. Acad. Sci. USA. 75,3813-3817.