[CANCER RESEARCH 46, 4665-4671. September 1986]

Differential Effects of Transforming Growth Factor-/? on Proliferation of Normal

and Malignant Rat Liver Epithelial Cells in CultureJames B. McMahon, William L. Richards, Anthony A. del Campo, Min-Kyung H. Song, and Snorri S. Thorgeirsson1

Laboratories of Experimental Therapeutics and Metabolism [J. B. M., A. A. d. C.] and Experimental Carcinogenesis [W. L. R., M-K. H. S., S. S. T.], National CancerInstitute, NIH, Bethesda, Maryland 20892

ABSTRACT

Transforming growth factors ( I (,!•-/is) have been shown to cause both

stimulatory and inhibitory effects on cellular growth in a variety of normaland neoplastic cells. The nature of the inhibitory effects of TGF-/3 onproliferation of different cell types is at present unclear. We have usedfreshly isolated rat hepatocytes, a normal diploid rat liver epithelial cellline (NRLM), and a subline (AFB) derived from it which was transformedin vitro by aflatoxin Bt to study the nature of TGF-0-induced growthinhibition and its alteration following chemically induced neoplastic transformation. TGF-/3 had a vastly different effect on proliferation of normalrat liver epithelial cells (both freshly isolated and NRLM cells) comparedto aflatoxin Bi-transformed cells. TGF-/3 at 20 pg/ml caused 83% inhibition of colony formation of NRLM, whereas the growth of AFB cellswas unaffected by TGF-/J at concentrations as high as 10 ng/ml. Aparallel dose-dependent inhibition of DNA synthesis by '!'(,!•'-/<was

observed in both primary hepatocytes and NRLM cells at concentrationsbetween 10 pg and 10 ng/ml. No inhibition of DNA synthesis wasobserved in AFB cells. Furthermore, TGF-/3 did neither induce anchorage-independent growth of NRLM cells nor affect the growth of AFBcells in soft agar. TGF-0-induced inhibition of the NRLM cells wasirreversible in nature, since treated cells were unable to proliferate andform colonies upon removal of TGF-/J from the medium. Also, NRLMcells showed, after 4 days in the presence of 20 pg of TGF-0 per mlmorphological changes characterized by cytoplasmic hypertrophy andthe formation of abundant liposomal derivatives, some of which resemblelipofuscin. The finding that TGF-/? caused a high degree of irreversibleinhibition of NRLM cells emphasizes the need for caution in interpretingdata from inhibition studies, since most assays presently used are designed for assessing growth stimulation in vitro and do not adequatelydistinguish between the possible cytotoxic and/or cytostatic action ofgrowth inhibitors.

INTRODUCTION

Characterization of factors that control cellular proliferationremains a central issue in the continued attempts to understandboth normal and neoplastic growth. Although the presentknowledge in this area is far from complete major progress hasbeen made in the last several years on the role of polypeptidegrowth factors in the control of cell proliferation. The availability of purified factors has made possible the elucidation ofsome of the normal regulatory mechanisms as well as providinginsight into alteration in both cellular replication and differentiation associated with neoplastic transformation. Althoughmost studies have dealt with polypeptide growth factors whichare growth stimulatory (for review, see Ref. l), several reportshave appeared that describe purified cell proliferation inhibitors(2-4).

One major subclass of polypeptide growth factors which hasreceived considerable attention recently is the family of growthfactors collectively termed TGFs2 (5, 6). TGFs, which promote

Received 1/17/86; revised 4/30/86; accepted 5/22/86.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' To whom requests for reprints should be addressed.2The abbreviations used are: TGF, transforming growth factor; EGF, epider

mal growth factor; TGF-«, transforming growth factor «;TGF-/Î,transforminggrowth factor -í:AFB¡.aflatoxin B,; HPI, hepatic proliferation inhibitor.

the anchorage-independent growth of anchorage-dependent fi-broblast cell lines, are subdivided into two categories based onthe capacity to compete with EGF for receptor binding (TGF-as) (7) and whether EGF (or TGF-as) are needed additionallyto promote growth of nontransformed fibroblasts in soft agar(TGF-/3S) (8-12). Both types of TGFs have been purified tohomogeneity from a variety of normal and neoplastic cells (10,13-17). Although much has already been learned about thestructure and biochemistry of TGFs with respect to their actionon nontransformed fibroblasts, very little has been reported onthe effects of these growth factors on nonmalignant epithelialcells. The need to study the effects of these growth factors onepithelial cells is underscored by a recent report where it wasshown that, under certain cellular conditions, TGF-0 can act asa potent inhibitor of cell proliferation in many types of cells,including both neoplastic and nonneoplastic cells of eitherfibroblastic or epithelial morphology (18). Furthermore, TGF-ßis both structurally and functionally related to an inhibitor ofepithelial cell proliferation isolated from conditioned mediumfrom African green monkey cells (BSC-1) (19). In the presentstudy we have utilized freshly isolated hepatocytes, a normaldiploid rat liver epithelial cell line, and a subline derived fromit which was transformed in vitro by aflatoxin BI to study thenature of TGF-/3-induced growth inhibition and its alterationfollowing chemically induced neoplastic transformation.

MATERIALS AND METHODS

The cell line of normal liver epithelial cells (NRLM) was establishedfrom a 10-day-old male Fischer (F344) rat by the methods of Herringet al. (20). For this study the NRLM line was used between the fifthand 10th passage in vitro. Cells were subcultured weekly and weremaintained as monolayer cultures. The transformed liver cell line (AFB)was established from the NRLM cells after they had been transformedin vitro by treatment with AFB,. Transformation was accomplished byexposure of low-passage NRLM cells with a nontoxic dose (50 ng/ml)of AFB], continuously for 10 wk, during which time the cell line wassubcultured weekly. The cells were tumorigenic when implanted s.c. inFischer rats 10 wk after the end of carcinogen treatment. Both cell lineswere maintained routinely in Ham's F-12 medium supplemented with

10% defined fetal bovine serum (Hyclone Laboratories, Logan, UT).The cells were grown on plastic culture dishes (Falcon Plastics, Oxnard,CA) and incubated at 37°Cin humidity cabinets with a gas phase of

5% CO2 in air.Primary hepatocytes were freshly isolated from adult (180 to 220 g)

male F344 rats by the two-step collagenase perfusion technique (21).The cells were cultured for 72-h in the above medium further supplemented with transferrin (5 Mg/ml) and dexamethasone (0.02 j<g/ml).The mitogenic stimulus for the primary hepatocytes used in this studywas EGF (20 ng/ml). Medium changes were at 3 h and 24 h afterplating.

Cellular proliferation was assayed by several methods. The methodused for measuring the effects of TGF-,3 on the extent of DNA synthesisand growth of normal and malignant rat liver cells was that of Song etal. (22) and Richards et al. (23). Briefly, single cell suspensions wereseeded in 96-well microtiter plates in a volume of 100 ¿ilper well.Twenty h after plating, medium was changed to culture medium containing various concentrations of TGF-/Î,and plates were incubated for

4665

on June 28, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECTS OF TGF-/3 ON RAT HEPATOCYTES

48 h at 37°Cin a humidified atmosphere of 5% CO2/95% air. Duringthis incubation, cells were labeled with [mefA.y/-3H]thymidine (0.5 mCi/

well) for the last 2 h in the case of established rat liver epithelial cellsand for 24 h with primary hepatocytes. The medium was removed, cellswere washed with serum-free F-12 medium, and 100 M' of serum-free

medium per well with Hoechst 33342 dye (8 Mg/ml) were added. Afterincubation for 30 min at 37"C, fluorescence in each well was read in

the MicroFLUOR reader (Dynatech Laboratories, Inc., Alexandria,VA). Trypsin solution (5% trypsin/0.6% NaCl solution/0.01 M sodiumcitrate, pH 7.4, 22 //I/well) was added, and plates were incubated at37°Cuntil cells had detached. Cells were collected on glass fiber filters

and lysed with distilled water, and the radioactivity of the cell chromatinwas determined by scintillation counting.

The biological activity on TGF-/3 was also determined by a quanti-

tative colony-forming assay based on the reversible inhibition of cell

proliferation by partially purified hepatic proliferative inhibitor. Thisassay, which has been described in detail elsewhere (24, 25), involvedplating cells at a colonial density (20 cells per cm2) in 60-mm plasticPetri dishes. After a 1-day attachment period, the cells were treated

with either control medium or control medium plus varying concentrations of growth factors and/or growth inhibitors. After 4 days, themedium was replaced with control medium, and the cells were maintained for an additional 5 days (recovery period). Cell colonies werethen fixed in methanol and stained with Giemsa. The total number andsize of the stained cell colonies were determined by using an ArtekModel 880 automatic colony counter (Artek Systems, Farmingdale,NY). The effect of TGF-ßon the anchorage-independent growth of the

liver epithelial cells was assessed by standard soft agar techniques.

Fig. 1. Photomicrographs of rat liver epithelial cells. A, phase-contrast photomicrograph of NRLM cells, x 280. B, phase-contrastphotomicrograph of AFB cells, x 280. C photomicrograph of a section of a tumor resultingfrom the s.c. injection of AFB cells. H & E, x150.

irI.'••-;

* * \, \ .: •"/>•.'*'

4666

on June 28, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECTS OF TGF-/3 ON RAT HEPATOCYTES

Fig. 2. Effect of TGF-ff (20 pg/ml) on morphology of NRLM cells after 4 days in culture.A, control NRLM cells. Giemsa stain, X 300.B, TGF-/3-treated NRLM cells. Giemsa stain,x 300. C, transmission electron microscopicimage of control NRLM monolayers. x 1000./>. transmission electron microscopic image ofTGF-fi-treated NRLM monolayers. x 1000.

i . -

Briefly, various concentrations of TGF-ßin F-12 culture mediumcontaining 10% fetal bovine serum, 0.3% agar, and 2 x IO4cells wereadded to 35-mm culture plates on top of a base layer of 0.5% agar inculture medium. Some plates received EGF at a final concentration of2 ng/ml. The plates were incubated at 37°Cin a humidified atmosphere

containing 95% air and 5% CO2. After 14 days the colonies werephotographed at low-power magnification.

Electron Microscopy. Control cells and cell treated with TGF-ßfor4 days were first washed 2 times with warm saline and subsequentlyfixed in situ with 2% glutaraldehyde in 0.1 M cacodylate buffer with 0.1M sucrose (osmolarity, 500 mOsmol, pH 7.2) for l h at room temperature. Cells were then post-fixed with 1% OsO4 in 0.1 M cacodylatebuffer with 0.1 M sucrose for 45 min at 4°C.This was followed by

dehydration in a graded series of alcohols and embedding in situ byinverting EPON-filled BEEM capsules over the monolayers. Polymerized blocks were removed from the culture dishes with pliers.

Tissue from transplanted tumors was minced into pieces and fixedby immersion with 2% glutaraldehyde in 0.1 M cacodylate buffer with0.175 M sucrose (550 mOsmol, pH 7.2) for 2 h at room temperature.Subsequent processing was identical to that for monolayer cells, exceptthat pieces of tissue were polymerized in BEEM capsules. Sections ofSO inn thickness were stained with uranyl acetate and lead citrate andexamined in a JEOL 100 CX electron microscope.

Morphometric Analysis. Semiconfluent monolayers received eithermedium alone (control) or medium plus TGF-0 (20 pg/ml) and werecultured for 4 days. Cells were then fixed in methanol and stained withGiemsa. Stained culture dishes were examined at x25 with a Zeiss lightmicroscope interfaced with a Zeiss Videoplan 2 image analysis system.After calibration with a stage micrometer, nuclear and cytoplasmic areameasurements were taken. A total of 100 cells from each group wasanalyzed. A standard / test was performed on the data.

RESULTS

The NRLM cell line contained cells with a uniform epithelialmorphology as seen by phase contrast (Fig. IA) and by electronmicroscopy (Fig. 2C). The cells had a low saturation density,and their proliferation was highly anchorage dependent (Fig.3/1). The NRLM cells contained a diploid number of chromosomes throughout the study and were not tumorigenic whentransplanted into F344 rats.

The AFB,-transformed NRLM cells (AFB cells) show considerable morphological similarities to the parent cells (Fig.IB). However, these cells differ greatly with respect to many of

4667

on June 28, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECTS OF TGF-,9 ON RAT HEPATOCYTES

Fig. 3. Growth of rat liver epithelial cellsafter 14 days in soft agar. A, NRLM cells; B,AFB cells in the presence of 20 pg of TGF-01per ml: C. AFB cells in the presence of 20 pgof TGF-f* per ml plus 2 ng of EGF per ml; D,AFB cells in 2 ng of EGF alone per ml. x 100.

B

the parameters associated with neoplastic cells. These includedability to proliferate under anchorage-independent conditions

(Fig. 3Ä),reach high saturation densities (Fig. I/?), and to formtumors on injection into syngeneic rats (Fig. 1C). The resultingtumors when examined by electron microscopy revealed areasof hepatocytes forming bile canaliculi lined by microvilli characteristic of hepatocellular carcinomas (Fig. 4).

Effect of TGF-/9 on the Proliferation of Normal and MalignantLiver Cells in Vitro. The effect of TGF-/3 on the proliferationof normal and malignant rat liver cells was first examined by a96-well microtiter plate assay. In this assay method, the extentof DNA synthesis (the incorporation of [3H]thymidine) wasnormalized by the cell number by dividing the 3H cpm value

with the DNA fluorescence unit. As shown in Fig. 5, DNAsynthetic activities in both primary hepatocytes and NRLMcells were inhibited in a dose-dependent fashion by TGF-/3 in

the final concentration range between 10 pg/ml and 10 ng/ml.The inhibitory effects on both NRLM cells and primary hepa

tocytes were parallel throughout the dose range tested. TGF-/3did not show any inhibitory action on rat liver epithelial cellstransformed with AFB,.

The inhibition of proliferation seen in the microtiter assaywas further examined by a colony-forming assay. The NRLMcells were found to be extremely sensitive to inhibition by TGF-ß.This inhibition was reflected both in the number and size ofthe cell colonies present after the TGF-/3 treatment. Fig. 6,left, shows that 20 pg of TGF-/3 per ml resulted in more than

83% inhibition of colony formation by the NRLM cells. Higherconcentrations of TGF-ßessentially abolished the colony formation of these cells. In sharp contrast to these results, TGF-/3had no significant inhibitory effect on the growth of the AFB|cells (Fig. 6, right). This difference in sensitivity of NRLM andAFB, cells to TGF-0 was further substantiated by light andelectron microscopic observations. The NRLM cells show, after4 days in the presence of 20 pg of TGF-/3 per ml, morphologicalchanges characterized by cytoplasmic hypertrophy (Fig. 25;

4668

on June 28, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

on June 28, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECTS OF TGF-ßON RAT HEPATOCYTES

cytoplasmic areas were considerably more variable in the TGF-/3-treated cells. This is reflected in the high standard deviationof the cytoplasmic area measurements (Table 1). None of thesechanges was observed in AFB cells following the same treatment with TGF-ß.

Effect of TGF-ßon Growth of NRLM and AFB Cells in SoftAgar. The NRLM cells did not form colonies in soft agar underany of the conditions tested (Fig. 3/4). The AFB cells whichformed colonies in soft agar (Fig. 3Ä)without added growthfactors (other than fetal bovine serum) were not affected byTGF-ß. A dramatic increase in the anchorage-independentgrowth of AFB cells was seen, however, when EGF (2 ng/ml)was added either alone (Fig. 3C) or in the presence of TGF-ß(20 pg/ml) (Fig. 3D).

DISCUSSION

TGF-ßs have been shown to cause both stimulatory andinhibitory effects on cellular growth in a variety of normal andneoplastic cells (18). It has also been shown that the epithelialcell growth inhibitor, isolated from conditioned medium ofAfrican green monkey kidney cells, is either identical to TGF-ßor belongs to the same family of peptides (19). Furthermore,TGF-/3 can exhibit bifunctional response (i.e., both stimulationand inhibition) in a single cell type (myc-1 rat fibroblasts) (18).In this paper we present evidence that TGF-ßhas a vastlydifferent effect on the proliferation of normal rat liver epithelialcells (both freshly isolated and NRLM cells) and on the in vitroAFB i-transformed cells derived from the NRLM cells (Figs. 5and 6). Concentration of TGF-0 as low as 20 pg/ml caused an83% inhibition of colony formation of NRLM cells, whereasthe growth of AFB cells is unaffected by concentrations ofTGF-ßas high as 10 ng/ml (Fig. 5). Furthermore, TGF-ßdoesneither induce anchorage-independent growth of NRLM cellsnor affect growth of AFB cells in soft agar (Fig. 3). The findingthat EGF alone caused a dramatic increase in the anchorage-independent growth of the AFB cells is noteworthy. This mayrepresent a new transformation-linked phenotype. How andwhen this phenotype is expressed during chemical transformation in vitro is currently under investigation.

The nature of the inhibitory effects of TGF-/3 on proliferationof different cell types is at present unclear. Roberts et al. (18)showed that TGF-ßcaused a dose-dependent increase in theapparent doubling time (as determined by cell counts) of neoplastic A-549 cells without affecting viability. No data werepresented to show if this effect was reversible or if all the cellsin the population were equally inhibited. From our data it isclear that the high degree of inhibition of the NRLM cellscaused by TGF-ßunder these conditions was irreversible innature; i.e., treated cells were unable to proliferate and formcolonies upon removal of TGF-0 from the medium. Since theprimary hepatocytes had only limited proliferative capabilitythe nature of the parallel inhibition that was observed withTGF-ßin the microtiter assay could not be accurately determined. The finding that TGF-ßcaused a high degree of irreversible inhibition to the normal epithelial liver cells emphasizesthe need for caution in interpreting data from inhibition studies,since most assays presently used are designed for assessinggrowth stimulation in vitro and do not adequately distinguishbetween the possible cytotoxic and/or cytostatic action ofgrowth inhibitors. Although the exact mechanism(s) of inhibition caused by TGF-ßon the nontransformed liver cells remainsunknown, observations at the ultrastructural level revealed thatTGF-ß caused an increase in lysosomal derivatives in the

NRLM cells. This phenomenon often represents an adaptiveresponse of the cytoplasm to an altered functional state (26).Moreover, the enlargement of cytoplasmic area without a parallel enlargement of the nuclei is an indicator of disturbancesof cellular control mechanisms similar to what has been reported for neoplastic epithelial cells (27). However, inhibitionof NRLM cell proliferation by either maintaining the cellscontact inhibited or by serum starvation (data not shown) didnot result in the cytoplasmic alterations similar to those seenafter TGF-ßtreatment or decreased colony formation. Whetheror not the ultrastructural changes are associated with a cytotoxic response to TGF-ßremains to be determined.

The lack of inhibitory activity TGF-ßto the NRLM cellsfollowing chemical transformation is similar to the loss ofresponsiveness of transformed rat liver epithelial cells to HPI(4). HPI is a Mt 26,000 protein that has been shown to inhibitthe proliferation of nonmalignant rat liver epithelial cells butdoes not inhibit the division of malignant liver cells, whetherthey are induced in vivo or in vitro (4, 24, 25, 28).

The inhibition of proliferation exerted by HPI is reversiblein that, upon its removal, the nonmalignant liver cells begindivision. This is in contrast to the mechanism(s) of inhibitionexerted by TGF-ßon these cells, because treatment under thesame experimental conditions appears to abolish their proliferative capability and leads to irreversible morphologicalchanges. Treatment of normal human bronchial epithelial cellswith TGF-ßresults in squamous differentiation characterizedby an increase in cell surface area, formation of cross-linkedenvelopes, and cessation of cell division (29), while malignanthuman lung carcinoma cells or bronchial epithelial cells transformed by v-Harvey ras p21 oncogene show no such responseto either inducer (30). The possibility therefore exists that TGF-ßis a differentiation-inducing agent for the NRLM cells. In

this context, both the morphological and functional changesobserved in the NRLM cells following TGF-ßtreatment mayreflect changes in the differentiation stage of these cells, perhapscloser to the fully differentiated adult hepatocyte. Work alongthese lines is presently in progress.

ACKNOWLEDGMENTS

The gift of pure TGF-ßfrom Dr. M. Sporn is gratefully acknowledged. We also thank Dr. H. M. Schuller for help with the electronmicroscopy studies.

REFERENCES

1. James, R., and Bradshaw, R. A. Polypeptide growth factors. Annu. Rev.Biochem., 53: 259-292, 1984.

2. Holley, R. W., Armour, R., Baldwin, J. H., and Greenfield, S. Activity of akidney epithelial cell growth inhibitor on lung and mammary cells. Cell Biol.Int. Rep., 7:141-147, 1983.

3. Holley, R. W., Bohlen, P., Fava, R., Baldwin, J. H., Kleeman, G., andArmour, R. Purification of kidney epithelial cell growth inhibitors. Proc.Nati. Acad. Sci. USA, 77: 5989-5992, 1980.

4. McMahon, J. B., Farrelly, J. G., and type, P. T. Purification and propertiesof a rat liver protein which specifically inhibits the proliferation of nonmalignant rat liver epithelial cells. Proc. Nati. Acad. Sci. USA, 79:456-460,1982.

5. DeLarco, J. E., and Todaro, G. J. Growth factors from murine sarcomavirus-transformed cells. Proc. Nati. Acad. Sci. USA, 75:4001-4005, 1978.

6. Todaro, G. J., DeLarco, J. E., Fryling, C, Johnson, P. A., and Sporn, M. B.Transforming growth factors (TFGs): properties and possible mechanismsof action. J. Supramol. Struct. Mol. Biochem., 15: 287-301, 1981.

7. Carpenter, G., Stoscheck, C. M., Preston, Y. A., and DeLarco, J. E. Antibodies to the epidermal growth factor receptor block the biological activitiesof sarcoma growth factor. Proc. Nati. Acad. Sci. USA, 80:5627-5630,1983.

8. Anzano, M. A., Roberts, A. B., Meyers, C. A., Komoriya, A., Lamb, L. C.,Smith, J. M., and Sporn, M. B. Synergistic interaction of two classes oftransforming growth from murine sarcoma cells. Cancer Res., 42: 4776-4778, 1982.

9. Anzano, M. A., Roberts, A. B., Smith, J. M., Sporn, M. B., and DeLarco, J.

4670

on June 28, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECTS OF TGF-/3 ON RAT HEPATOCYTES

E. Sarcoma growth factor from conditioned medium of virally transformedcells is composed of both type a and ßtransforming growth factors. Proc.Nati. Acad. Sci. USA, 80:6264-6268. 1983.

10. Assoian, R. K., Komoriya, A., Meyers, C. A., and Sporn, M. B. Transforminggrowth fauni ,; in human platelets: identification of a major storage site,purification, and characterization. J. Biol. Chem.. 258: 7155-7160, 1983.

11. Roberts, A. B., Anzano, M. A., Lamb, L. C, Smith, J. M., Frolik, C. A.,Marquardt, II. Todaro, G. J., and Sporn, M. B. Isolation from murinesarcoma cells of novel transforming growth factors potentiated by EGF.Nature (Lond.), 295:417-419, 1982.

12. Roberts, A. B., Anzano, M. A., Lamb, L. C., Smith, J. M., and Sporn, M.B. New class of transforming growth factors potentiated by epidermal growthfactors: isolation from non-neoplastic tissues. Proc. Nati. Acad. Sci. USA,78: 5339-5343, 1981.

13. Frolik. C. A., Dart. L. L., Meyers, C. A., Smith, D. M.. and Sporn, M. B.Purification and initial characterization of a type ft transforming growthfactor from human placenta. Proc. Nati. Acad. Sci. USA, 80: 3676-3680.1983.

14. Marquardt. H., Hunkapiller, M. W., Hood, L. E., and Todaro, G. J. Rattransforming growth factor type 1: structure and relation to epidermal growthfactor. Science (Wash. DC), 223: 1079-1082, 1984.

15. Nickell, K. A., Halper, J., and Moses. H. L. Transforming growth factors insolid human malignant neoplasms. Cancer Res., 43: 1966-1971, 1983.

16. Roberts, A. B., Anzano, M. A., Meyers, C. A., Wideman, J., Blacher, R.,Pan, Y-C. E., Stein, S., Lehrman, S. R., Smith, J. M., Lamb, L. C., andSporn, M. B. Purification and properties of type ßtransforming growthfactor from bovine kidney. Biochemistry, 22: 5692-5698, 1983.

17. Salomon, D. A., Zwiebel, J. A.. Bano, M., Losonezy, !.. Fehnel, P., andKidwell, W. R. Presence of transforming growth factors in human breastcancer cells. Cancer Res., 44:4069-4077, 1984.

18. Roberts, A. B., Anzano, M. A.. Wakefield, L. M., Roche. N. S., Stern, D.F., and Sporn, M. B. Type ßtransforming growth factor: a bifunctionalregulator of cellular growth. Proc. Nati. Acad. Sci. USA. 82: 119-123, 1985.

19. Tucker, R. F., Shipley, G. D., Moses, H. L., and Holley, R. W. Growthinhibitor from BSC-I cells closely related to platelet type ßtransforming

growth factor. Science (Wash. DC), 226: 705-707, 1984.20. Herring, A. S., Raychaudhuri, R.. Kelley, S. P., and lype, P. T. Repeated

establishment of diploid epithelial cell culture from normal and partiallyhepatectomized rats. In Vitro (Rockville), 19: 576-588, 1983.

21. Evarts, R. P., Marsden, E., Hanna. P., Wirth. P. J., and Thorgeirsson, S. S.Isolation of preneoplastic rat liver cells by centrifugal elutriation and bindingto asialofetuin. Cancer Res., 44: 5718-5724, 1984.

22. Song, M-K. H., Krutzsch, H., Hankins, W. D., Richards, W. L., and Thorgeirsson, S. S. Rapid determination of DNA synthesis in adherent cellsgrown in microtiter plates. Exp. Cell Res., 156: 271-276, 1985.

23. Richards, W. L., Song, M-K., Krutzsch, H., Evarts, R. P., Marsden, E., andThorgeirsson, S. S. Measurement of cell proliferation in microculture usingHoechst 33342 for the rapid semiautomaled microfluorimetric determinationof chromatin DNA. Exp. Cell Res.. 159: 235-246, 1985.

24. lype, P. T., and McMahon. J. B. Hepatic proliferation inhibitor. Mol. CellBiochem., 59: 57-80, 1984.

25. McMahon, J. B., and lype, P. T. Specific inhibition of proliferation of non-malignant rat hepatic cells by a factor from rat liver. Cancer Res., 40: 1249-1254, 1980.

26. Krstic, R. V. (ed.). Ultrastruktur der Saugetierzelle, pp. 116-121. Berlin:Springer-Verlag, 1976.

27. Reznik-Schuller, H. M., and Haque. B. F., Jr. A morphometric study of thepulmonary Clara cell in normal and nitrosohepatamethyleneimine-treatedEuropean hamsters. Exp. Pathol., /*: 366-371, 1980.

28. lype, P. T., and McMahon, J. B. Lack of correlation between (he responseto a proliferation inhibitor and other transformation markers in a mutantliver cell line. Cancer Res., 41: 3352-3354, 1981.

29. Masui. T., Wakefield, L. M., Lechner, J. F., LaVeck, M. A., Sporn, M. B.,and Harris, C. C. Type ßtransforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells.Proc. Nati. Acad. Sci. USA, 83: 2438-2442, 1986.

30. Yoakum, G. H., Lecher, J. F., Gabrielson. E., Korba, B. E., Malan-Shibley,L., Willey, J. C., Valerio, M. G., Shamsuddin, A. K. M., Trump, B. F., andHarris, C. C. Transformation of human bronchial epithelial cells transfectedby Harvey ras oncogene. Science (Wash. DC), 227: 1174-1179, 1985.

4671

on June 28, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1986;46:4665-4671. Cancer Res   James B. McMahon, William L. Richards, Anthony A. del Campo, et al.   in CultureProliferation of Normal and Malignant Rat Liver Epithelial Cells

onβDifferential Effects of Transforming Growth Factor-

  Updated version

  http://cancerres.aacrjournals.org/content/46/9/4665

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/46/9/4665To request permission to re-use all or part of this article, use this link

on June 28, 2018. © 1986 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from