[CANCER RESEARCH 47, 5600-5605, November 1, 1987]

Extraction and Partial Purification of a Hepatic Stimulatory Substance in Rats,Mice, and Dogs1

Antonio Francavilla,2 Peter Ove, Lorenzo Polimeno, Mona Coetzee, Leonard Makowka, John Rose,

David H. Van Thiel, and Thomas E. StarzlDepartment of Gastroenterology, University of Bari, Bari, Italy [A. F., L. P.J, and Departments of Anatomy and Cell Biology [P. O., M. C.J, Surgery [L. M., T. E. S.J,and Gastroenterology [D. H. V. T.¡,University Health Center of Pittsburgh, University of Pittsburgh, Pittsburgh 15261, and Department of Crystallography, VeteransAdministration Medical Center [J. R.J, Pittsburgh, Pennsylvania 15240

ABSTRACT

A factor has been isolated from weanling rat liver which stimulates invivo hepatic DNA synthesis in a dose dependent manner when injectedinto 40% hepatectomized rats. The factor has been partially purified bysuccessive steps, involving ethanol precipitation, ultrafiltration throughan Amicon PM 30 membrane, and finally fast protein liquid chromatog-raphy, resulting in a 38,000-fold increase in specific activity over that inthe original cytosol. The factor contains a few bands in the molecularweight range of 14,000-50,000 on sodium dodecyl sulfate-polyacrylamide

gel electrophoresis.Active fractions from fast protein liquid chromatography (Fist), when

injected into 40% hepatectomized rats, increased hepatic DNA synthesis3-fold over the background stimulation due to the hepatectomy. Theresponse was dose dependent over a range from 1.76 /tg to 6.8 ¿tgper200-g (body weight) rat. Mitotic and labeling indexes confirmed that l-'m,

stimulates both replicative DNA synthesis and cell proliferation. Thefactor is heat and neuraminidase resistant, trypsin sensitive, organ specific, but not species specific.

INTRODUCTION

Since the demonstration by Higgins and Anderson (1) of theremarkable capacity of the liver to regenerate, following surgicalremoval of 70% of the tissue, many investigators have attempted to elucidate the mechanism(s) that triggers the regenerative response. The use of the parabiotic model in rats yieldedevidence that the regenerative stimulus, which initiates andmaintains DNA synthesis and cell division, is transmitted viathe circulation (2). However, the nature of the humoral factorsinvolved is still undefined, despite the impetus provided to theirstudy by the use of hepatocytes in primary cultures (3-8).

Regenerating liver or weanling rat liver have been investigated as primary sources of hepatomitogens. In addition, avariety of hormones and defined growth factors have beenshown to modulate the regenerative response in vivo or tostimulate DNA synthesis in hepatocytes in primary culture.Among these are insulin and glucagon (9), EGF3 (3. 9), proline(10, 11), norepinephrine (12-14), and platelet derived growth

factors (15, 16).A number of investigators have isolated factors from serum

of PH rats capable of stimulating DNA synthesis of hepatocytesin primary culture (17-20). However, these preparations havenot been shown, thus far, to be active in vivo. There are also anumber of reports on the isolation and partial purification of

Received 11/19/86; revised 4/1/87, 7/21/87; accepted 7/31/87.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.

1This study was supported by a research project grant from the VeteransAdministration, Project Grant AM-29961 from the NIH, Bethesda, MD, andGrant 885/02 16544 from Consiglio Nazionale delle Ricerche, Italy.

2To whom requests for reprints should be addressed, at Veterans Administration Hospital, Building 6, Circle Drive. Pittsburgh, PA 15240.

'The abbreviations used are: EGF, epidermal growth factor; PH, partiallyhepatectomized; HSS, hepatocyte stimulating substance; FPLC, fast proteinliquid chromatography; PBS, phosphate buffered saline; SDS, sodium dodecylsulfate.

substances from regenerating rat, dog, or rabbit livers or fromweanling rat liver (19, 21-30). Notably the presence of a HSSin regenerating and weanling rat liver was first reported byLaBrecque and Pesch (21). We have previously reported thatcrude preparations from proliferating liver tissues stimulatedDNA synthesis in vivo (27, 28). In this report we describe theisolation and purification of a factor from weanling rat liverwhich stimulates DNA synthesis in vivo in a dose dependentmanner.

MATERIALS AND METHODS

Animals

Male Fischer (F344) rats (180-200 g) and weanling rats (60-90 g),female CF-1 mice, and male mongrel dogs (15-20 kg) were purchasedfrom Hilltop Lab Animals, Scottsdale, PA, and were kept in temperature and light controlled rooms. They received food and water adlibitum.

Surgical Procedures

Partial hepatectomies of either 40% or 70% were performed in allanimals between 7:30 and 9 a.m. Hepatectomies were performed in ratsand mice according to the method of Higgins and Anderson (1) and indogs as described previously (23). Control animals underwent a shamoperation consisting of laparotomy and manual manipulation of theliver.

Materials

Neuraminidase type V and proteins used as molecular weight markers were purchased from Sigma Chemical Company, St. Louis, MO.[methyl3H]Thymidine (50-80 Ci/mmol), '"I-EGF (200 MCi/Mg),and'"I-vasopressin (1820 jiCi/^g) were purchased from New EnglandNuclear, Boston. MA. '"Mnsulin (100 /¿Ci/Mg)and 125I-glucagon(200fiCi/Mg) were purchased from Amersham-Searle Corp., ArlingtonHeights. IL, and L-l-tosylamido-2-phenylethyl chloromethyl ketone-trypsin was purchased from Worthington Biochemical Corp., Bedford,MA. Amicon ultrafiltration membrane filters were purchased fromAmicon Corporation, Danvers, MA.

Preparation of Hepatic Extracts

Cytosols

Cytosols were prepared from livers of normal rats, from regeneratingliver remnants 24 h after 70% PH, and livers of weanling unoperatedrats. The livers were excised, placed in 4 volumes (w/v) of ice-coldbuffer (0.27 M sucrose-12 mM Tris-HCl-1 mM EDTA, pH 7.6), andhomogenized, using a Potter-Elvenhjem tissue grinder. The homoge-nates were centrifuged for 10 min at 10,000 x g and 4°C,and the

supernatant was again centrifuged for 1 h at 100,000 x g using a Spincoultracentrifuge.

HSS Preparation and Purification

Ethanol Precipitation Fraction. This fraction (OH-F) was preparedessentially as described by LaBrecque et al. (21, 25, 26) with slightmodification. The livers from sham-operated rats, weanling unoperatedrats, 70% PH rats, or 70% PH dogs were homogenized in cold 100

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HSS PURIFICATION, HEPATIC STIMULATION IN VIVO

mM sodium acetate, pH 4.65 (35% w/v), between 7 and 8 a.m. A pHof between 4 and 5 was determined to be necessary for the most efficientextraction of HSS. Homogenates were heated at 65°Cfor 15 min andcentrifuged at 30,000 x g for 20 min at 4°C.Six columns of cold

ethanol were added to the supernatants. After being stirred for 2 h at4'C the supernatants were centrifuged at 30,000 x g for 30 min at 4°C.

The resulting pellet was dissolved in 150 mM ammonium acetate,lyophilized, and stored at —70°Cuntil use. The amount of HSS obtained

in the ethanol precipitate was identical whether the homogenate washeated directly or the heating step was applied to the cytosol. Forinjection into animals, the lyophilized OH-F was dissolved in 5 mMphosphate buffer, pH 7.4.

Amicon Membrane Ultrafiltration. Lyophilized OH-F, dissolved in150 mM ammonium acetate, or freshly prepared OH-F in 150 mMammonium acetate was filtered through Amicon PM 30 ultrafiltrationmembranes. The PM 30 filtrate was then concentrated, using AmiconYC05 membranes with a molecular weight limit of 500. The retainedconcentrated solution was designated as the M, 30,000 fraction. Thisfraction was lyophilized and stored at —70°C.

Fast Protein Liquid Chromatography. The lyophilized A/, 30,000fraction (20 mg) was suspended in 5 mM phosphate buffer, pH 6, andchromatpgraphed on a Mono Q HR 5/5 column, using ABS-751 FPLCapparatus (Pharmacia, Uppsala, Sweden). Elution of material wasachieved with a linear 0-200 mM NaCl gradient in 5 mM phosphatebuffer, pH 6, at a flow rate of 2 ml/min. UV absorbance peaks (280nm) were collected and dialyzed against 150 mM ammonium acetate,lyophilized, and stored at —70°Cuntil use. The fractions were dissolved

in 5 mM phosphate buffer, pH 7.4, and, after the determination ofprotein, tested for stimulatory activity. Some additional absorbancematerial could be eluted with 500 mM NaCl, but no protein was detectedin this fraction. Sixteen mg of total protein (80%) were recovered,including the void volume.

Analyses of Physical and Chemical Properties

Active fractions eluted from the Mono Q column were tested fortrypsin sensitivity; aliquots (3 pg) were dissolved in distilled water,brought to pH 7.6, and incubated at 37"C with L-l-tosylamido-2-

phenylethyl chloromethyl ketone-trypsin (100 /ug/ml). After 2 h thereaction was stopped by addition of 2 ¿igof soybean trypsin inhibitor/jig of trypsin. A mixture of similar amounts of trypsin and inhibitorwas preincubated for 30 min at 20°Cbefore addition to the active

fraction and was similarly treated as a control (31). To test for heatstability, aliquots (3 fig) were dissolved in PBS, pH 7.6, and heated at95*C in boiling water for 10 min. Neuraminidase sensitivity (19) was

tested on aliquots (3 /¿g)dissolved in water, adjusted to pH 5.5, andincubated at 37°Cfor l h in the presence of 0.5 unit of neuraminidase/ml. The reaction was terminated by heating at 95'C for 30 min; after

centrifugation the supernatant was used for i.p. injections. As a control,0.5 unit of neuraminidase, without any HSS present, was treated thesame way and injected.

SDS-Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide gradient slab gels, 7.5 to 20% with a 5% stacking gel, were prepared and developed according to the method ofLaemmli (32). Protein bands were visualized by Coomassie Blue R 250according to the method of Weber and Osborn (33).

Protein Determination

Protein was determined by the method of Lowry et al. (34) or by themethod of McKnight (35) for the determination of submicrogramquantities.

Determination of the Activity of HSS and Its Fractions

Fraction activity was determined in vivo using rats and mice. Experiments were carried out according to the method of LaBrecque andPesch (21). Briefly a heightened background of DNA synthetic activityin vivo was induced in host rats and mice by a 40% PH. The 40%hepatectomized animal model was chosen for its sensitivity to recognizeeither an inhibitor or a stimulatory factor. Six h after PH the rats were

given i.p. injections of 2 ml of 5 mM phosphate buffer, pH 7.4 (PBS-control), cytosol, ethanol precipitation fraction (OH-F), M, 30,000fraction, and the active FPLC fraction (Fiso), at protein concentrationsas indicated in the tables. Seventeen h later, 50 ¿iCi[3Hjthymidine were

injected i.p., and the animals were sacrificed l h later.Extracts (0.2 nil volume) were also administered i.p. to mice at 30 h

after 40% PH and DNA synthesis was studied 18 h later. [3H]Thymidine

(10/iCi/mouse) was injected i.p. l h before sacrifice (i.e., 47 h followingPH). Nonhepatectomized rats received injections of extracts 24 and 18h before determination of [3H]thymidine incorporation, and mice received injections at 48, 24, and 18 h. [3H]Thymidine incorporation,

labeling, and mitotic indexes were determined as described previously(23). An augmentation of all 3 parameters, beyond the modest responsethat is usually present in 40% PH or in unoperated animals, wasconsidered to be indicative of a proliferative inducing activity of theliver extracts.

DNA Synthesis Determination in Organs Other Than the Liver

DNA synthesis in the small intestine, spleen, heart, and kidney wasalso determined in 40% PH rats, following injection of Fl5o, and wascompared to DNA synthesis observed in the same organs of 40% PHrats given injections of PBS.

Statistical Analysis

Statistical analysis of groups were made by one way analysis ofvariance using SPSS/PC statistical software (SPSS, Inc., Chicago, IL)on an IBM-AT microcomputer.

RESULTS

Determination of HSS Activity in Cytosol. Five ml of cytosol(75 mg of total protein) of weanling rat liver or regeneratingrat liver remnants 24 h after 70% PH stimulated hepatic DNAsynthesis when injected into recipient rats with a 40% PH. Theresults shown in Fig. 1 represent at least 5 separate experiments.A 40% PH alone resulted in a 2- to 3-fold increase in hepaticthymidine incorporation when compared to control sham-operated nonhepatectomized rats. Injection of cytosol preparedfrom normal adult rat liver did not result in a further stimulation. However, cytosol prepared from regenerating rat liver orfrom weanling rat liver produced a further stimulation of 3-fold(P < 0.05). Since the same degree of stimulation was achievedwith either source, weanling rat liver was used in all subsequentexperiments for purification of HSS. Since there was no differ-

100T

«„80 Ho o

60 -

(!) Q

If 40

> è.c at- o

i-i ~ 20

n

Fig. l. DNA synthesis in sham-operated and 40% hepalectomized rats afterthe i.p. injection of rat liver cytosol. DNA synthesis in the liver was determinedas described under "Materials and Methods." H. sham-operated rats given injec

tions of PBS; Q. 40% hepatectomy plus PBS; D, 40% hepatectomy plus cytosolfrom normal rat liver, •,40% hepatectomy plus cytosol from 24-h regeneratingrat liver, G. 40% hepatectomy plus cytosol from weanling rat liver. Columns,means from 20 rats; bars, SD. *, significantly different from the control value (P< 0.05).

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HSS PURIFICATION, HEPATIC STIMULATION /AT VIVO

enee in liver DNA synthesis between rats given injections of 5HIM PBS and those injected with cytosol prepared from thelivers of normal nonhepatectomized animals, phosphate bufferwas used as controls in all subsequent experiments. Nonhepatectomized recipient rats did not respond with an increasedhepatic DNA synthetic activity when given injections of cytosol,regardless of the source, i.e., regenerating or nonregenerating(results not shown). Thus, an augmentation of hepatic regeneration by cytosol prepared from proliferating liver cells couldbe elicited only in a heightened background of hepatic DNAsynthesis produced by 40% PH.

Determination of HSS Activity in Ethanol Precipitate. Thefirst step of cytosol purification by ethanol precipitation produced a 7.5-fold enrichment factor (Table 1). OH-F extractedfrom weanling rat liver or from regenerating rat liver 24 h after70% PH stimulated hepatic incorporation of tritiated thymidineby 4-fold with respect to the animals given injections of OH-Fprepared from sham-operated rats.

Determination of HSS Activity of M, 30,000 Fraction. Furtherpurification (4-fold) was achieved by ultrafiltration on Amiconmembranes and yielded the M, 30,000 fraction. One-fourth theamount of protein of this M, fraction resulted in the samestimulation of DNA synthesis as did 10 mg of OH-F. Theseresults are shown in Fig. 2. Also shown in Fig. 2 are the mitoticand the labeling indexes confirming that OH-F and the M,30,000 fraction stimulate both replicative DNA synthesis andcell proliferation.

Table I Effect of OH-F prepared from different sources on DNA synthesis,percentage of labeled nuclei, and percentage of mitosis in 40%

hepatectomized ratsThe preparation of ethanol precipitate fraction (OH-F), determination of I'll]

thymidine incorporation, labeling index, and mitotic index have been describedin "Materials and Methods." Rats received 10 mg of OH-F i.p. in 2 ml S mM

phosphate buffer.

Source ofOH-FSham-operated

ratliver (control)

70% PH rat liverWeanling rat liverNo.

ofrats814

16[JH]thymidine

incorporation(cpm/mgDNA)13,130

±690°63.520

±18,100*60,750 ±13,320*%of

labelednuclei12.8

±1.8

25.8 ±5"%of

mitosis0.7

±0.1

1.8 ±0.2*

°Mean ±SD.* Significantly different from sham-operated rat liver (control value); P < 0.05.

T, » 4° "I

» 0)

= > 30 H(U y•°° on 4co ** «O_i <"

# » 10Hi o J

- 1 e»o

iL-0 *

midine>»fnZ

no<«0O

0)Q^".-^.oEü

U.£°uu

-SO

-vjW10-ni0)E

o1*

JL,o>Eevi

Control OH-F 30Kd

Fig. 2. Effect of the ethanol precipitate fraction and M, 30,000 fraction (30Kd) on DNA synthesis and proliferation. Experimental conditions are describedunder "Materials and Methods." Columns, average of 30 rats; bars, SD. *,

significantly different (P < 0.05).

Determination of HSS Activity in FPLC Fraction. Applicationof the M, 30,000 fraction to a FPLC column resulted in afurther substantial purification and a 38,000-fold increase inspecific activity. The elution profile in a linear 0-200 HIMNaCIgradient in 5 mM phosphate buffer, pH 6, and the activity ofthe various fractions is shown in Fig. 3. The activity of thesefractions was evaluated in 40% PH rats and expressed as cpm/fig DNA/Vg of protein injected. Although several of the elutionpeaks demonstrated some activity, the material eluting at 150HIMNaCI produced the greatest stimulation. This fraction wasdesignated F,50.

The activity of this fraction was dose dependent over a rangeof 1.76 to 6.8 Mg/rat with an average weight of 200 g. [3H]

Thymidine incorporation results were confirmed by labelingand mitotic index results, as shown in Fig. 4.

The administration of OH-F, M, 30,000 fraction, or Fi50 intosham-operated rats, as already demonstrated with cytosol, didnot result in stimulation of DNA synthesis. Even multipleinjections of these stimulatory fractions did not augment DNAsynthesis activity in sham-operated rats (data not reported).

The degree of purification of the activity achieved thus far isreported in Table 2. In calculating the purification, the originalcytosol was used as the starting material. Ethanol precipitationresulted in only modest purification as did ultrafiltration on anAmicon membrane filter with a molecular weight limit exclusion of 30,000. The purification was significantly improved byfractionation with FPLC, resulting in a 38,000-fold increase inthe specific activity of Fi50 over that in the original cytosol. Itmust be noted that injection of these extracts into non-PH ratsand mice under the conditions reported in "Materials andMethods" was ineffective (data not shown). The activity in F,50was resistant to neuraminidase and to heating at 95°for 10 min

but was trypsin sensitive.To rule out the possibility that the stimulatory activity exhib

ited by the fractions in these studies could be due to the presenceof hormones known to stimulate hepatic DNA synthesis in vivoor in vitro, I25l-labeled vasopressin, insulin, glucagon, and EGF

s§5'*•o—Z

r-i o E

40-

20-

EeO00CM

CO!n

1.61

08-

0.2-

B

150 200 mM NaCI

Fig. 3. Elution and activity profile of HSS from FPLC. Stimulatory activityof FPLC fraction (A). Elution profile of 30 Kd (B). The amount of protein injectedfor each fraction was 3 jig except for F150of which only 1.5 ^g were used for thisexperiment. [3H]Thymidine incorporation in rats given injections of 3 *ig of

bovine serum albumin was 5378 ±690 cpm//ig DNA//jg protein. Each was anaverage from 6 different animals. The statistical analysis shows that only thevalue of F,»was significant (P < 0.0001 ).

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HSS PURIFICATION, HEPATIC STIMULATION IN VIVO

DOSE RESPONSE CURVE 2

wUÃŽ

o

iif

—<DQJ —ID (J

CO D

3

2

1

0

30

20

10

0

100

I--"

illto D

<o oí 50a =•

, « E1 o ae o

0J

0.0 1.7 3.4 6.8

Fig. 4. Dose-response curve in 40% hepatectomized rats given injections ofFiso. The experimental conditions are the same as those reported in Fig. I.Control animals (10) were given injections of PBS containing 6.6 Mgof serumalbumin. DNA synthesis, percentage of labeled nuclei, and percentage of mitosiswere done as reported in "Materials and Methods." Points, averages of 10determinations for each level of F150;bars, SD. *, significantly different from thecontrol value (PBS) (P < 0.05).

Table 2 Steps of purification of HSS and chemical and physical properties offraction F150obtained from weanling rat liver

The purifie.il »HIscheme of HSS has been described in "Materials and Methods." The determination of [3H]thymidine incorporation was as for Table 1. The['Hlthymidine incorporation in a 40% hepatectomized rat given an injection of

PBS was 16,550 ±3.000 cpm/mg DNA. The numbers are the averages from noless than 20 different rats ±SD.

MaterialinjectedCytosol

65°CsupernatantOH-FM, 30.000 fractionFPLC F,»Protein

in- Specific activityjected/rat DNA synthesis (units/mg Fold of

(mg) (cpm/mg DNA) protein)purification75

2010.52.70.00343.350

±8,82056,720 ±10,24066.350 ±11.35063,520 ±13,22054,380 ±10,2000.02

0.120.301.05

762.06

15102

38,100

were mixed with M, 30,000 fraction and passed through theFPLC Mono Q column. All counts were eluted with less than50 mM NaCl concentrations, well before the major active peakwhich eluted at 150 mM NaCl (data not shown). It has beenshown previously that the in vitro activity of HSS is not due tocontamination with these or other well known hormones (26).

The results of SDS-polyacrylamide gel electrophoresis areillustrated in Fig. 5; ethanol precipitate, M, 30,000 fraction,and F,5o were compared. F,,,, still contained several bands, withmolecular weights ranging from 14,000 to 50,000.

Organ and Species Specific Activity Study. Fi50 was used todetermine the organ specificity of HSS, as shown in Fig. 6.Injection of Fi50 into 40% PH recipient rats resulted in asignificant stimulation of DNA synthesis only in the liver (P <0.001), without affecting DNA synthesis of the heart, kidney,spleen, and small intestine.

The results listed in Table 3 indicate that F150activity is notspecies specific. Indeed, F,50 prepared from weanling rats stimulated DNA synthesis in 40% PH female CF-1 mice, while FISO

205

66

4536

2924

2O

14.2

Fig. 5. SDS-polyacrylamide gel electrophoresis of different purification stepsof HSS. The gel electrophoresis preparation is reported in "Materials and Methods." Slot I, M, 30,000 fraction: slot 2, fraction OH-F; slot 3, fraction F,»;slot

4, a mixture of protein standards (Sigma) with molecular weights multiplied byio-3.

80-1

70-

zo6°-EE

S50-c0«

40-k-n00a^S

30-0)CÜ

20-E

(~i—

-i 10-

I>•0

*

I I li ver

Heart

Spleen

| Kidney

t ' :'| Small Intestine

40% Hepatectomy+ FISO

40% HepatectomyControl

Fig. 6. Specificity of F15ofor hepatic DNA synthesis. Weanling rat liver F,«was injected into a 40% hepatectomized rat. DNA synthesis was determined inthe liver, the heart, small intestine, kidney, and spleen, as described in "Materialsand Methods." Columns, averages from 5 rats; bars, SD. *, significantly different

(P < 0.001 ) from the value observed in the liver of the control animals.

prepared from canine liver remnants at 48 h after 70% PHsignificantly stimulated DNA synthesis in 40% PH rats.

DISCUSSION

The purpose of this study was to purify and characterizeHSS, a protein found in the liver which has been reported to

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HSS PURIFICATION, HEPATIC STIMULATION IN VIVO

stimulate hepatic DNA synthesis and hepatocyte replicationand which was previously identified by LaBrecque et al. (21,25, 26), by Francavilla et al. (27, 28) in rat liver, and by Starzlet al. (23, 24) in the canine liver. Despite the numerous attempts, a true liver specific mitogen has not been purified toany great extent. LaBrecque, who first used the term hepatocytestimulatory substance, in recent abstracts (36, 37) has reporteda significant purification of HSS. In our study the evaluationof proliferating activity of various fractions was carried out inan in vivo model using 40% PH rats. This model has beendemonstrated by us (27, 28, 38) and by others (21, 25, 26) tobe the most sensitive and reproducible assay system for detecting the presence of activity in such studies.

Physicochemical studies of the FISOfraction demonstratedthat HSS seems to be a protein with a molecular weight between50,000 and 14,000, which is resistant to neuraminidase, isdestroyed by trypsin, and is resistant to heating at 95°Cfor 10

min. Each of the preparations obtained from FPLC chromatog-raphy was completely free of recognizable hormones such asinsulin, glucagon, vasopressin, and EGF. Specifically, insulinand glucagon are soluble in alcohol, while HSS is precipitatedin alcohol. Furthermore, these hormones, labeled with I25I,

eluted from a Mono Q column in a position completely differentfrom that of F,50 (data not shown).

Table 3 Species specificity offlsa obtained from liver of yeanling rats and 70%hepatectomized dogs and injected into mice or rats

A 40% PH was performed on the recipient mice and rats. Injections and DNAdetermination with rats were done as described in Fig. 4. Mice were giveninjections of the indicated amounts of I•,.„in :i volume of 0.2 ml 30 h after 40%PH. DNA synthesis was determined after a 1-h exposure at 48 h after theoperation. Values are the averages from 6 animals ±SD.

Source andamount ofFISO(Mg)PBSRat

O.SRat1.0Dog

3DogòDog 9[3H]Thymidine

incorporation (cpm/mgDNA) in recipientanimalsMice7,250

±1,02516,650

±2,055°25,750 ±6,500"Rats16,550

±3,00024,250

±4,875"32, 175 ±9,875°41,750± 10,300"

' Significantly different from the control value (PBS) (P < 0.05).

Table 4 reports all the attempts made since 1957 to isolateor prove the presence of a specific growth factor in the liver ofdifferent species. In all cases, the factors isolated are reportedto be organ specific and species unspecific, all were preparedfrom rat liver homogenate, and in only one case (39) was itisolated from cellular membrane.

Concerning the physicochemical characteristics, all factorswere sensitive to trypsin, but not all had heat stability, whilethe molecular weight ranged between 14,000 and 45,000.Among all these reports, only 3, including ours, address theproblem of purification.

At present it is difficult to compare the HSS preparation ofLaBrecque and our preparation. Although many chemical andphysical characteristics are similar, there are substantial differences in the activity. For example, our HSS stimulates DNAsynthesis in vivo only in animals with a 40% hepatectomy andthe stimulation is dose dependent. LaBrecque and Bachur (25)have reported that a crude HSS preparation stimulates hepaticDNA synthesis in normal rats and mice but have not testedtheir newest preparation (40) in vivo or with hepatocytes inprimary culture. Their primary assay system has been an HTCculture system. We have not been able to confirm that our HSSstimulates DNA synthesis in HTC cells. This might be due todifferences in HTC lines maintained in different laboratories.Since neither our HSS nor the HSS of LaBrecque is completelypure, a final comparison is possible only after complete purification.

It is important to note that factors which have been able tostimulate hepatocyte regeneration have been found in the perfusate of isolated and partial hepatectomized livers of rats anddogs, confirming the possibility of the passage of this factorinto the circulation. Serum factors, in fact, have recently beenfound by several authors (15-20, 29, 31, 41, 42).

Although the final identification and purification of the specific growth factor(s) derived from the liver remain to be defined, our data represent a significant advancement. A highdegree of purification has been achieved and it has been clearlydemonstrated that F150is active only on hepatocytes exhibitinga dose-response phenomenon by both DNA synthesis and percentage of mitosis. The fraction, as it presently exists, stillcontains a few bands on SDS polyacrylamide gel electrophoresis

Table 4 Summary of theliteratureInvestigatorBlomqvistLaBrecqueHalaseStarzlGoldbergTerblancheFrancavillaSchwarzLiebermanFleigTime

period19571975-1987197919791980-198519801984-1985198519841986BiologicalName of

sourcesubstanceNbReL"WRL-ReRL

HSSNLReL

from70%HedogReL

from 70%Hepato-HeratpoietinRe

dogLWRLHSSReL

from70%HeRandpigMouse

plasmamembraneReL

from60%HerabbitBiological

assaysystemin

vivo: NRinvifro:HTCcellsinvivo: 40%HeRinvitro:L-929fibroblastIB

vivo: N and34%HeRin

vivo: dogwithportacavalshuntm

vivo:NRIB

vivo:NRIBvivo: 40%HeRIBvivo: 34% He female R andWRin

vitro:hepatocytecellsin

vi'fro: NR-6linefibroblastIB

vivo: NRPhysicochemical

characteristics:(resistance)HeatYesNoYesYesYesNoTrypsin

ChymotrypsinNeuraminidaseNo

NoYesNoNo

NoNoNo

NoYesYes

NoNoNo

NoResponse

or- % ofgan/species purificationW,"Yes

No 110,000 14,000- 15,00030,000YesYes

No 13,00038,000Yes

No 38,00015,000-50,000No14,000-25,000

°L, liver, N, normal; Nb, newborn; R, rat; Re, regenerating; He, hepatectomized; W, weanling.* Determined by SDS-polyacrylamide gel electrophoresis

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HSS PURIFICATION, HEPATIC STIMULATION IN VIVO

and it therefore cannot be ruled out that some of them mightcontain inhibitory activity.

The potential of these contaminating proteins may also bethe reason for the need to use 40% hepatectomized rats in ourassay system. Once complete purification has been achieved itis conceivable that HSS can exhibit more potent activity whichwill allow a better definition of the role of HSS in the chain ofthe liver regeneration. Similarly a completely pure HSS mightstimulate DNA synthesis of hepatocytes in primary culture.

Apart from the biological implications, the exact definitionof HSS and other hepatic growth factors has important clinicalimplications. The use of growth factor therapy for acute liverfailure in animals and in humans is, in fact, the main objectiveof this study. We have already shown that this type of therapy,using fractions obtained during the HSS purification, improvesthe survival rate of rats intoxicated with the selective hepato-toxin D-galactosamine (43).

ACKNOWLEDGMENTS

We are grateful to John Prelich for technical assistance.

REFERENCES

1. Higgins, G. M., and Anderson, R. M. Restoration of the liver of the whiterat following partial surgical removal. Arch. Pathol., 12:186-202, 1931.

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