Functional diversity of gro gene expression in human fibroblasts and ...

Proc. Natl. Acad. Sci. USA Vol. 85, pp. 9645-9649, December 1988

Genetics

Functional diversity of gro gene expression in human fibroblasts and mammary epithelial cells (tumors/interleukin 1/phorbol 12-myristate 13-acetate/gene mapping)

ANTHONY ANISOWICZ*, DEBORAH ZAJCHOWSKI*, GORAN STENMANt, AND RUTH SAGER*t *Division of Cancer Genetics, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; and tDepartment of Oral Pathology, Gothenburg University, S-400 33 Goteborg, Sweden

Contributed by Ruth Sager, August 26, 1988

leukin 1 (IL-1) induces at least a 100-fold elevation. The responses to serum and PMA of c-myc and c-fos mRNAs are also similar to those reported as growth-induced in other systems (14, 15), whereas IL-1 elicits no change in c-myc or c-fos gene expression. These results suggest that the growthinduced and IL-1-induced regulatory pathways are distinct in fibroblasts and that gro is involved in both modes. The gro gene is also expressed in normal mammary fibroblasts and epithelial cells. However, gro mRNA is absent or greatly reduced in most carcinoma cell lines examined including those from lung and colon as well as breast, whereas it is expressed in melanomas, which are mesenchymal rather than epithelial in origin. These varied responses raise the possibility that gro cDNA may be detecting more than one gene. However, by in situ hybridization of gro cDNA to the human chromosome complement, we find only a single site located at chromosome 4q21, in agreement with Richmond et al. (6). Similarly, in CHEF/18 cells, only a single gro hybridizing site is present, located at chromosome lpS.

ABSTRACT Previous studies of gro and related genes that are overexpressed in transformed fibroblasts suggest that gro may encode a specific growth regulator. However, DNA and protein sequence comparisons reveal relatedness to platelet factor 4 and other proteins involved in the inflammatory response. In this paper, both growth-related and cytokineinduced responses in gro gene expression are described. Human foreskin fibroblasts are shown to express 10-fold elevated gro, myc, and fos mRNAs in response to serum and to phorbol 12-myristate 13-acetate stimulation, with early response kinetics indicative of growth regulation. In response to interleukin 1, however, in growing cells gro mRNA is elevated at least 100-fold but myc remains constant and fos is not expressed, suggesting a second regulatory pathway. In normal cultured mammary epithelial cells, gro is constitutively expressed, and elevated mRNA levels are induced by phorbol 12-myristate 13-acetate, but not by interleukin 1. However, most carcinoma cell lines examined do not express gro mRNA, suggesting a third function of gro as a negative growth regulator in epithelial cells.

The gro gene is constitutively overexpressed in the transformed Chinese hamster fibroblast cell line CHEF/16 but is under tight regulatory control in the closely related nontransformed CHEF/18 cells (1). Comparison of gro cDNAs from Chinese hamster and human cells showed high sequence conservation (1). Sequence similarity was also noted with a cDNA overexpressed in chicken fibroblasts transformed by v-src (2, 3). Another structurally related cDNA (called KC) is a platelet-derived growth factor-inducible gene turned on during growth of BALB/c 3T3 fibroblasts (4, 5). More recently, a human gene with the same cDNA sequence as that of human gro has been shown to encode a secreted protein that stimulates the growth of a melanoma cell line (6). These reports suggest that gro may be a ubiquitous growth factor. Sequence comparisons of DNA and protein in GenBank have shown that gro is a member of a family of genes encoding secretory proteins associated with the inflammatory response. These include 3-thromboglobulin (7), connective tissue activating peptide III (8), platelet factor 4 (9, 10), and y-interferon-activated peptide (11). Another related cDNA was found in lymphotoxin-activated leukocytes (12), and more recently a related protein probably encoded by this cDNA was described as a chemotactic agent for neutrophils (13). These sequence relationships suggest that gro may also have a function in the inflammatory response. In this paper, we describe three distinct modes of response of the gro gene in a variety of cell types. We report that human foreskin fibroblasts express a 10-fold elevated steadystate level of gro mRNA in response to serum or phorbol 12-myristate 13-acetate (PMA) stimulation, whereas inter-

MATERIALS AND METHODS Cells and Growth Conditions. The FS-2 cell strain derived from foreskin fibroblasts as described (16) was grown in alpha-MEM (Hazelton Systems, Vienna, VA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 2 mg of glucose per ml, insulin (10 ,ug/ml), 1% human serum (GIBCO), 2 mM glutamine, penicillin (100 units/ml), streptomycin (100 ,ug/ml) in humidified 6.5% C02/93.5% air at 37°C and p$f 7.3. Tumor cell lines other than the breast lines were grown in this medium without added glucose, insulin, or human serum. The normal human mammary epithelial strains 184 and 172, derived from mammoplasty tissues by Stampfer et al. (17), and 30N, derived in this laboratory, were grown in DFCI-1 medium (18). The tumor cell lines grown in this laboratory and harvested in late logarithmic-phase growth for RNA isolation were as follows: RD (rhabdomyosarcoma), Kaposi sarcoma, T24 (bladder carcinoma); CX-1, CCL218, and CCL228 (three colon carcinomas); CCL 185, OAT 4, A549, SCC25, and 23A (five lung carcinomas); CRL 1435 (prostatic carcinoma); HUTU 80 (duodenal carcinoma); CRL 1420 (pancreatic carcinoma); A2058, G361, and SKMF30 (three melanomas); MCF-7, ZR-75-1, BT-20, T47D, MDAMB231, and HS578T (six mammary carcinomas). Preparation of DNA and RNA. Total RNA was prepared by the guanidium/CsCl method (19) from subconfluent cells. Northern blots were prepared and hybridized as described (1); f-actin hybridization was used to verify equal loading of RNA. Autoradiograms were scanned with a Quick Scan R&D densitometer (Helena Laboratories, Beaumont, TX). Appro-

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Abbreviations: PMA, phorbol 12-myristate 13-acetate; IL-1, interleukin 1. tTo whom reprint requests should be addressed.

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Genetics: Anisowicz et al.

priate exposures were used to give signals in the linear range of the film and densitometer. Probes. The following probes were used: for gro, the 721-base-pair Nco I/BamHI fragment of human gro cDNA (1); for myc, the 1.4-kilobase (kb) Cla IlEcoRI fragment from the third exon of human genomic myc (pMYC BL22) (20); for fos, the 1-kb Pst I/Pvu II fragment from v-fos (pFBJ-2) (21); for actin, the 2-kb chicken f3-actin cDNA excised with Pst I from pAl (22). All probes were 32P-radiolabeled to 1-3 x 109 cpm/lg (1). Growth Kinetics. For serum stimulation, cells were pregrown with medium containing 0.5% serum for 48 hr. For PMA and IL-1 stimulation, cells in midlogarithmic growth phase were treated with PMA (100 ng/ml) (Sigma) or recombinant human IL-1 (2.5 units/ml) (Genzyme, Norwalk, CT) in alpha-MEM salts for the time indicated. In Situ Chromosomal Hybridization. Human and Chinese hamster gro cDNA probes were radiolabeled by nicktranslation as whole plasmids with [3H]dTTP to specific activities of 3-8 107 cpm per ,ug of DNA. The labeled probes were hybridized to normal human metaphase chromosomes from two individuals (one male and one female) and to chromosomes from early passage (nos. 2-5) primary Chinese hamster embryo cells (obtained from P. M. Kraemer, Los Alamos, NM) as described (23, 24). The hybridizations were carried out at probe concentrations of 10-100 ng/ml hybridization solution and with a 1000-fold excess of carrier herring sperm DNA. Washes after hybridization were at 39°C-420C. After being coated with NTB2 photographic emulsion (Kodak) and exposed for 10-20 days, the autoradiographs were developed and G-banded using Wright stain (25). The chromosomal positions of grains located on chromosomes or touching chromosomes were recorded on ideograms of G-banded human (26) and Chinese hamster chromosomes (27).

mRNA in FS-2 cells compared with CHEF/18 cells is primarily the result of message stability. These results indicate a substantial difference between FS-2 and CHEF/18 cells in posttranscriptional regulation of gro mRNA levels. In contrast, the rapid induction and disappearance of fos mRNA is similar in the two cell types (Fig. 2a; ref. 1). In growing FS-2 cells to which the tumor promoter PMA was added (Fig. 2b), gro mRNA was elevated above the untreated steady-state level. The PMA-induced gro mRNA was diminished somewhat by 4 hr, in contrast to the stability of the serum-induced mRNA. The c-myc mRNA peaked at 1 hr, and that for c-fos peaked at 30 min. Thus, PMA elicited a transcriptional response by all three genes, similar to the serum-induced growth response. In a search for evidence of gro involvement in the inflammatory response as suggested by the related superfamily of gro-like genes and proteins, we examined the response of gro expression to treatment with the cytokine IL-1 (28). As shown in Fig. 2c, gro mRNA expression in FS-2 cells was induced >100-fold by IL-1 added at zero time to exponentially growing cells. c-myc mRNA was already high at zero

RESULTS

cells. In contrast, the carcinoma-derived MCF-7 cells did not express gro when grown with either alpha-MEM or with DFCI-1 medium (lanes 3 and 5). The two media were compared to ascertain whether the differences in serum content or other factors influenced gro expression. To determine whether MCF-7 cells could express gro mRNA under any circumstances, we inhibited protein synthesis with cycloheximide, a process that elevates gro message =30-fold in CHEF/18 cells (1). Cycloheximide treatment elevated gro mRNA -10-fold in 184 cells (lane 2) and elicited a weak but reproducible response in MCF-7 cells (lane 6). Thus, the absence of gro expression in MCF-7 cells may result from inhibition by a short-lived regulatory protein. Fig. 3 Lower shows the responses of 184 and MCF-7 cells to PMA induction. gro mRNA was elevated within 15 min in 184 cells and remained high for at least 6 hr. c-myc was somewhat elevated in 15 min and was gone by 2 hr. The fos response was biphasic with a dip at 1 hr and elevated expression at 0.5 and 2 hr. In contrast, no gro message was expressed in MCF-7 cells, although myc and fos messages were elevated. None of the mammary epithelial cells tested, whether normal or tumor-derived, responded to IL-1 by increased expression of gro, myc, or fos mRNAs (data not

X

Expression of gro mRNA in Human Fibroblasts. The tight regulation of gro mRNA levels in CHEF/18 cells results from a very low run-on transcription rate (1), as well as from mRNA instability as demonstrated in Fig. la. In contrast, human foreskin fibroblasts (FS-2 cells) in response to serum after serum-starvation (Fig. 2a) express a long-lived gro message, which is stable in the presence of actinomycin D (Fig. lb). Treatment with actinomycin D blocks further transcription, so that the observed mRNA levels show persistence of preexisting message. In a series of comparative experiments (data not shown), we found that the steady-state level of gro mRNA expression in FS-2 cells was 5- to 10-fold higher than in CHEF/18 cells (1), but the nuclear run-on transcription rate of FS-2 cells was similar to that of CHEF/18 cells. Thus, the elevated steady-state level of gro b

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FIG. 1. Analysis of stability of gro message. Subconfluent cells fed with fresh medium containing actinomycin D (5 pg/ml) and treated for the indicated time (hours). Total RNA (20 pug per lane) was electrophoresed (see Materials and Methods), transferred to nitrocellulose, and hybridized to 32P-labeled gro probe. (a) CHEF/18. (b) FS-2. Exposure times: CHEF/18, 8 days; FS-2, 2 days.

were

time. In similar experiments, the initial myc level was low but, at most, only a 2-fold increase was seen. Nofos message was detected at any time. Thus, the gro transcriptional response to IL-1 in FS-2 cells was quite different from the response to serum and to PMA. gro Expression in Epithelial Cells. The normal human mammary epithelial cell strain 184 (17) grow exponentially on

plastic in a medium, DFCI-1, recently developed in our laboratory (18). In other studies (29), these cells have been compared with a number of mammary carcinoma cell lines for expression of several growth-related genes. Here we compare the expression of gro mRNA in normal and tumorderived mammary epithelial cells. As shown in Fig. 3 Upper (lane 1), gro mRNA was expressed in exponentially growing normal 184 cells. This steady-state level is comparable to that observed in FS-2

shown). The steady-state level of gro mRNA was examined in a series of mammary tumor cell lines (Table 1) and in a series of human tumor cell lines from other tissues (Fig. 4). As shown in Table 1, gro was expressed only by mammary lines that contain an activated ras gene. A similar effect of the ras gene was shown in our previous paper (1), in which T24, a mutated c-Ha-ras-containing line, and FS-2 cells transfected

with mutated c-Ha-ras or infected with Kirsten murine sarcoma virus expressed elevated gro mRNA. We grew a



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FIG. 2. Time course of serum, PMA, and IL-1 stimulation of gro, mye, and fos mRNAs in FS-2 cells. Total RNA (20 ,ug per lane) from FS-2 cells was electrophoresed, transferred to nitrocellulose, and hybridized sequentially to 32P-labeled gro, myc, andfos probes. Autoradiograms were exposed 20 hr except IL-1/gro (6 hr) and IL-1/fos (3 days). Numbers are times of treatment in hours. (a) Serum stimulation of serum-starved cells. (b) PMA stimulation of growing cells. (c) IL-1 stimulation of growing cells.

number of other tumor cell lines and selected those in which cells grew well in logarithmic-phase cultures for extraction of 1

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FIG. 3. Cycloheximide (CHX) and PMA stimulation of 184 and MCF-7 cells. Northern blots (20 ug per lane) were prepared as described in Fig. 1. (Upper) CHX stimulation of gro mRNA. Total RNA from 184 (lanes 1 and 2) and MCF-7 (lanes 3-6) was hybridized to 32P-labeled gro probe. CHX (10 j.g/ml) was added to growing cultures (lanes 2, 4, and 6) for 3 hr. 184 and MCF-7 (lanes 5 and 6) were grown in DFCI-1 medium; MCF-7 (lanes 3 and 4) was grown in alpha-MEM. Autoradiogram was exposed 16 hr. Numbers on right are kb. (Lower) Time course of gro, myc, and fos mRNA expression in PMA-treated 184 and MCF-7 cells. Northern blots were prepared as described in Fig. 1, with total RNA from cells treated with PMA (100 ng/ml) for the indicated time (hours). Autoradiograms were exposed for 20 hr and hybridized sequentially to 32P-labeled gro, myc, and fos probes.

mRNA. The results (Fig. 4) show that the majority of tumor lines did not make appreciable amounts of gro mRNA during exponential growth. The cell lines that strongly overexpressed gro were T24 (lane C), the bladder carcinoma line known to contain activated Ha-ras; CCL185 (lane G), a lung carcinoma; and one melanoma line (lane 0). In those lines overexpressing gro other than T24, the presence of an activated ras or other interacting oncogene is not known. Chromosomal Localization of Human and Chinese Hamster gro Gene. Mapping of the human gro gene revealed a single locus on chromosome 4. A total of 82 cells were scored of which 42 (51%) had grains on chromosome 4. Specific labeling occurred over the proximal part of 4q with a distinct peak at band q21 (Fig. 5a). Forty-six percent of the grains on the long arm clustered at this band, representing 14% (19/139) of total grains. No other chromosomal site was labeled above background. The gro locus is therefore assigned to human chromosome 4, band q21, located within the region containing PF4 (32). In situ hybridization of gro to Chinese hamster chromosomes showed a significant accumulation of silver grains on the short arm of chromosome 1. Of a total of 102 grains from 80 cells examined, 54 (53%) were on chromosome 1. The largest accumulation of grains was noted over the midregion of the short arm. Forty percent of the grains on lp clustered Table 1. Expression of gro mRNA in normal mammary epithelial cells and tumor-derived cell lines Estrogen Mutant-ras gro mRNA Cells receptor Normal + 184 + 172 + 30 Tumor lines + MCF-7 + ZR-75-1 BT-20 + T47D + -+ MDA-MB231 + -+t HS578T *Ref. 30. tRef. 31.

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FIG. 4. Steady-state gro mRNA expression in tumor cell lines. Total RNA (20 ,ug per lane) was prepared from tumor cells, and Northern blots were hybridized to 32P-labeled gro probe. Autoradiogram was exposed for 20 hr. Lanes: A, RD; B, Kaposi sarcoma; C, T24, D, CX-1; E, CCL 218; F, CCL 228; G, CCL 185; H, OAT 4; I, 23A; J, A549; K, SCC 25; L, CRL 1435; M, HUTU 80; N, CRL 1420; 0, A2058, P, G361, Q, SKME 30.

at band p5 (Fig. 5b), representing 19% (19/102) of total

chromosomal grains. The distribution of grains on all other chromosomes was random. We thus assign the gro locus to Chinese hamster chromosome 1, band p5.

DISCUSSION belongs to a superfamily of genes involved in the inflammatory response as well as in cell growth. Although other genes in this family have been associated with either cell growth (2-6) or inflammation (7-13), we show here that human fibroblasts respond to growth stimulation by serum and by PMA and to the cytokine IL-1 with elevated levels of gro gene expression. The early response kinetics of gro mRNA expression in serurh-stimulated or PMA-treated fibroblasts, which also express elevated myc and fos mRNAs, is strong but indirect evidence for gro involvement in growth regulation. The extent of the IL-1-induced response is at least 100-fold above the uninduced level, whereas the serum and PMA-induced levels are only -10-fold elevated. In addition, gro, myc, and fos mRNAs show coordinate responses to serum and to PMA, whereas neither myc or fos mRNAs are responsive to IL-1. These data support the hypothesis that the IL-1 response represents a distinct pathway involving gro but not myc orfos expression. Lin and Vilcek (38) described a rapid and transient increase in fos and myc mRNA levels in confluent serum-starved human fibroblasts in response to IL-1. Their experiment differs from ours in that we used exponentially growing cells. By this means, we dissociated the special growth response of serum-starved cells entering G1 from Go from the IL-1-

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FIG. 5. In situ hybridization. Silver grain distributions on human chromosome 4 (a) and Chinese hamster chromosome 1 (b) after in situ hybridization to 3H-labeled gro cDNA probes. The human gro locus is assigned to chromosome band 4q21 and the Chinese hamster locus is assigned to band lpS.

induced responses of growing cells. It should be noted that fos mRNA was elevated by PMA treatment of growing cells, further indicating a difference in PMA and IL-1 response pathways. To pursue this thesis, we turned to an examination of gro gene expression in IL-1-treated chondrocytes and synovial cells. Major functions of IL-1 are induction of collagen synthesis, collagenase formation, and other activities associated with wound healing and tissue remodeling (33, 34). Since Castor (8) had reported a role for the gro-related gene CTAP III in these processes, it was of interest to determine whether gro itself was involved. In preliminary experiments, we found that IL-1 induced a high level of gro mRNA in human rheumatoid synovial cells and costal chondrocytes. The untreated cells expressed little if any gro mRNA, whereas cells treated with IL-1 expressed high levels of gro message (R.S., M. B. Goldring, S. M. Krane, and A.A., unpublished data). These results support the hypothesis that gro plays a role in IL-1-induced events that may not be directly coupled to growth. The presence of two gro transcripts, 0.9 and 1.2 kb, was previously noted in CHEF/18 cells (1) and is now found in human fibroblasts and epithelial cells as well. In the studies described here, the 1.2-kb transcript was always present, usually as the major form. Expression of the 0.9-kb transcript was not seen in cycloheximide-induced mRNAs or in transcripts from IL-1-treated cells. However, it was present in serum-stimulated and PMA-treated cells, although not induced by PMA. Preliminary studies using fragments of gro cDNA as probes indicate that sequences in the 3' region of the gene are missing in the 0.9-kb band. It is possible that 3' sequences such as those conserved between the Chinese hamster and human gro (1) are involved in regulation of lower band expression. A totally unexpected finding in our survey of gro expression in various cell types was the presence of gro mRNA in normal growing mammary cells and its absence in the majority of carcinomas examined. The normal epithelial cells resembled fibroblasts in constitutive steady-state gro expression levels as well as in the levels of myc and fos mRNA in both untreated and PMA-treated growing cells. However, most of the carcinoma cell lines expressed little or no gro mRNA in exponentially growing cell populations. A detailed study with MCF-7, a mammary carcinoma cell line, showed that PMA treatment induced myc and fos expression but not gro, and, furthermore, that gro could be slightly induced by cycloheximide treatment. Thus, these tumor cells are capable of expressing gro, but are inhibited at the mRNA level, possibly by a short-lived negative regulatory protein. Lack of expression in tumor cells of a growth regulatory gene expressed in normal cells is one characteristic of tumor suppressor genes (35, 36). Thus, gro may have tumor suppressor activity in epithelial cells. However, evidence based on its overexpression in tumorigenic CHEF/16 cells (1), src-transformed chicken fibroblasts (2, 3), and human melanomas (figure 5, lanes O-Q in ref. 6) suggests that gro may be a positive growth factor for fibroblasts. Since gro is secreted (1, 4), its action may be autocrine or paracrine, affecting similar or different cell types as do other peptide growth factors. gro may resemble type p transforming growth factor (TGF-P) in stimulating growth of some cell types and inhibiting others (37). However, TGF-,B shows little regulation at the transcriptional level, whereas transcriptional regulation appears to be an important feature of gro function. If the gro gene is regulated by both positive and negative cis-acting elements, then differential regulation in different cell types would not be surprising. In summary, the results described in this paper implicate the gro gene in a variety of important cellular functions: as a putative position early response gene in cell growth, as a mediator of the IL-1-induced inflammatory response in


fibroblasts, and as a negative regulatory factor in epithelial cells. The evidence to date is largely indirect, and further studies with gro protein and antibody preparations should clarify these relationships. We thank Stephanie Budd and Dorothy Marden for preparing the manuscript, and Drs. L. B. Chen, S. Bernal, M. Wick, and F. Li for providing the tumor cell lines used in Fig. 5. This work was supported by National Institutes of Health Grant CA 39814 to R.S. and grants from the Swedish Medical Research Council (Project 7964) and the Swedish Cancer Society to G.S. 1. Anisowicz, A., Bardwell, L. & Sager, R. (1987) Proc. Natl. Acad. Sci. USA 84, 7188-7192. 2. Sugano, S., Stoeckle, M. Y. & Hanafusa, H. (1987) Cell 49, 321-328. 3. Bedard, P. A., Alcorta, D., Simmons, D. C., Luke, K.C. & Erickson, R.A. (1987) Proc. Natl. Acad. Sci. USA 84, 67156719. 4. Cochran, B. H., Reffel, A. C. & Stiles, C. D. (1983) Cell 33, 939-947. 5. Almendral, J. M., Commer, D., MacDonald-Bravo, H., Burckhardt, J., Perera, J. & Bravo, R. (1988) Mol. Cell. Biol. 8, 2140-2148. 6. Richmond, A., Balentien, E., Thomas, H. G., Flaggs, G., Barton, D. E., Spiess, J., Bordoni, R., Francke, U. & Derynck, R. (1988) EMBO J. 7, 2025-2033. 7. Begg, G. S., Pepper, D. S., Chesterman, C. N. & Morgan, F. J. (1978) Biochemistry 17, 1739-1744. 8. Castor, C. W., Miller, J. W. & Walz, D. A. (1983) Proc. Natl. Acad. Sci. USA 80, 765-769. 9. Deuel, T. F., Keim, P. S., Farmer, M. & Heinrikson, R. L. (1977) Proc. Natl. Acad. Sci. USA 74, 2256-2258. 10. Poncz, M., Surrey, S., LaRocco, P., Weiss, M. J., Rappaport, E. F., Conway, T. M. & Schwarz, E. (1987) Blood 69, 219-223. 11. Luster, A. D., Unkeless, J. C. & Ravetch, J. V. (1985) Nature (London) 315, 672-676. 12. Schmid, J. & Weissmann, C. (1987) J. Immunol. 139, 250-256. 13. Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E. A., Apella, E., Oppenheim, J. J. & Leonard, E. J. (1987) Proc. Natl. Acad. Sci. USA 84, 9233-9237. 14. Greenberg, M. E. & Ziff, E. B. (1984) Nature (London) 311, 433-437. 15. Kelly, K., Cochran, B. H., Stiles, C. D. & Leder, P. (1983) Cell 35, 603-610. 16. Sager, R. (1984) Cancer Cell 2, 487-493.


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