Cardnogeuesis vol.13 no.8 pp. 1481-1483, 1992
SHORT COMMUNICATION
Association between expression of transforming growth factor-alpha and progression of hepatocellular foci to neoplasms
William K.Kaufmann, Yingchun Zhang and David G.Kaufman Department of Pathology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA
Transforming growth factor-alpha (TGF-alpha*) is a growth factor for hepatocytes that is produced by proliferating hepatocytes in regenerating livers (1) as well as by a variety of tumor cells including a rat hepatocellular carcinoma line (2). Urinary TGF-alpha has been proposed as a tumor marker for management of patients with hepatocellular carcinoma (3). Analysis of phenotypic alterations that co-segregate with tumorigenesis in a liver epithelial cell culture model of carcinogenesis indicated that expression of TGF-alpha and acquisition of an autocrine growth cycle were positively correlated with tumorigenicity (4). We have used a commercial monoclonal antibody that was raised against human TGF-alpha to test for alterations in expression of TGF-alpha in rat hepatocellular neoplasms. This antibody preparation had been used to demonstrate elevated expression of TGF-alpha in renal carcinomas that
•Abbreviations: TGF-alpha, transforming growth factor-alpha; BPDE, (±)-7r,8»-dihydroxy-9r,10»-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; DMN-OAc, methyl(acetoxymethyl)nitrosamine; GSTP, glutathione-S-transferase (placental isoform). © Oxford University Press
Sections from the right lateral lobe that were free of neoplasms were processed for quantitation of altered hepatocellular foci. Serial paraffin-embedded sections of 5 /im were cut and stained for expression of glutathione-S-transferase (placental isoform) (GSTP) and TGF-alpha. Immunohistochemical staining for GSTP was performed according to the method described in the StrAviGen FASTest Universal Immunostaining Kit purchased from BioGenex Laboratories, San Ramon, CA. Anti-GSTP polyclonal antibody was kindly provided by Dr Ross Cameron, Toronto General Hospital, Toronto, Canada. A mouse monoclonal antibody to human TGF-alpha was obtained from Table I. Incidence of hepatocellular neoplasms that expressed TGF-alpha
Adenomas
DMN-OAc Carcinomas
BPDE Adenomas
Carcinomas
14/17" 82%
18/18 100%
11/13 85%
23/23 100%
"One TGF-alpha-negative adenoma was a GSTP-negative amphophilic adenoma.
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Hepatocardnogenesis was initiated in rats with a single dose of either of two chemical mutagens—benzo[a]pyrene diolepoxide I and methyl(acetoxymethyl)nitrosamine—administered 15 h after partial hepatectomy. Hie development of hepatocellular foci and neoplasms was then promoted with dietary phenobarbital given for 45 or 62 weeks. Formalin-fixed tissue specimens that contained hepatic neoplasms and altered hepatocellular foci were screened for expression of the oncodevelopmental marker glutathione-S-transferase (placental form) (GSTP) and transforming growth factoralpha (TGF-alpha) using immunohistochemistry. All (100%) hepatocellular carcinomas expressed both GSTP and TGFalpha, as did most hepatocellular adenomas (>80%). However, quantitative stereologic analysis of treated and control livers revealed that GSTP-positive foci were 10-30 tunes more frequent than TGF-alpha-positive foci. Foci with homogeneous expression of GSTP generally displayed heterogeneous expression of TGF-alpha with reaction product most prominent at their peripheries. Less frequently homogeneous TGF-alpha-positive foci were seen within GSTP-positive foci. The average volumes of those GSTPpositive foci that also expressed TGF-alpha were significantly greater than those of the entire sets of GSTP-positive foci. These results suggest that expression of TGF-alpha may distinguish a subset of GSTP-positive foci that have a growth advantage and increased probability of progression to neoplasia.
develop in a hereditary rat model (5). The current analysis revealed a high frequency of expression of TGF-alpha in hepatocellular neoplasms. However, when foci of hepatocellular alteration were examined, these lesions expressed TGF-alpha less frequendy and those that did usually displayed a heterogeneous staining pattern. The results suggest that acquisition of an autocrine growth cycle involving TGF-alpha may be an uncommon change in hepatocellular foci that heralds an increased probability of progression to neoplasia. Details of treatment of rats with (±)-7r,8/-dihydroxy-9r,10repoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and methyl (acetoxymethyl)nitrosamine (DMN-OAc) were as described previously (6,7). BPDE was purchased from Midwest Research Institute, Kansas City, MO. DMN-OAc was provided by Dr Jerry Rice, Frederick Cancer Research and Development Facility. Phenobarbital was from Sigma Chemical Co., Kansas City, MO. The diet ATN-76A with and without 0.05% phenobarbital was obtained from Zeigler Bros. Inc., Gardners, PA. F344 male rats were obtained from Charles River Breeding Laboratories, Raleigh, NC, at 6 weeks of age. One week after receipt rats were subjected to a two-thirds partial hepatectomy. Fifteen hours later BPDE and DMN-OAc were administered directly into the portal vein at a dosage of 0.02 and 0.1 mmol/kg respectively. Three weeks after treatment with BPDE and DMN-OAc (or after partial hepatectomy for controls), phenobarbital was added to the diet at 0.05%. Rats were fed this diet until 1 week before they were killed, when phenobarbital was withdrawn. At 45 and 62 weeks after initiation, rats were killed by exsanguination when under ether anesthesia. Livers were fixed in 10% buffered formalin, and then sectioned at 1 - 2 mm intervals. Visible tumor masses > 1 mm in diameter were identified and saved for paraffin embedding and histologic diagnosis in H&E-stained sections. Histopathological criteria for diagnosis of hepatocellular adenoma and carcinoma were as suggested by Maronpot et al. (8).
TGF-alpha in rat hepatocarcinogenesis
Table II. Incidences, numbers and volumes of GSTP-positive and TGF-alpha-positive hepatocellular foci Control
Incidence No./cm 2 No./cm 3 Mean volume ( x 10" 3 cm3)
GSTP
TGF-alpha
DMN-OAc GSTP
8/8 3.3 ± 2.4 247 ± 139 0.2 ± 0.1
1/7 0.4 ± l.l c 24 ± 64C 0.1 ± 0.1
5/5 18 ± 4" 487 ± 89" 2.3 ± 1.4"
TGF-alpha 5/5*
2± r 33 ± 31C 16.8 ± 10.8 bc
BPDE GSTP
TGF-alpha
9/9 42 ± 23" 1732 ± 1165" 1.4 ± 0.9"
9/9" 4 ± 2"-c 55 ± 3l"' c 9.5 ± 7.8"-c
Rats were given a single administration of DMN-OAc or BPDE at 15 h after partial hepatectomy followed by dietary exposure to phenobarbital. Controls received a partial hepatectomy and then dietary phenobarbital as for the carcinogen-treated groups. Livers were harvested 45 weeks after partial hepatectomy (controls) or 45—62 weeks after carcinogen treatment and processed for lmmunohistochemical demonstration of TGF-alpha and GSTP. Quantitation of foci as number/cm3 was done by quantitative stereology as described by Pugh el al. (9). 'Significantly different from control (chi-squared test, P < 0.05). Incidence is the number of positive livers divided by the number examined. "Significantly different from control (Student's t-test, P < 0.05). c Significantly different from GSTP (Student's (-test, P < 0.05).
Fig. 1. Expression of TGF-alpha and GSTP in hepatocellular foci. (A) Hepatocellular focus showing heterogeneous expression of TGF-alpha with prominent reaction product at periphery adjacent to collecting veins; (B) serial section containing the focus in (A) stained for GSTP; ( Q focus of more homogeneous expression of TGF-alpha; (D) serial section containing the focus in (C) stained for GSTP. Photomicrographs were made at 400x magnification. All sections were counter-stained with hematoxylin.
Oncogene Science, Manhasset, NY. After incubation of the antibody with paraffin sections for 18 h, visualization of antiTGF-alpha antibody binding was done using the FASTest kit. We reproduced the control observation of Walker ex al. (5) that preincubation of anti-TGF-alpha antibody with human recombinant TGF-alpha abolished immunohistcchemical reaction product over neoplasms and foci, whereas preincubation of the primary antibody with epidermal growth factor did not. Altered hepatocellular foci were quantified according to the method of Pugh et al. (9). Briefly, using a stereomicroscope equipped with a camera lucida, focal outlines were traced onto a digitizing tablet. A computer software program (BIOQUANT System IV, R&M Biometrics, Inc., Nashville, TN) calculated the area of each focus and the radius was determined assuming foci were spheroids. The numerical density of foci (number/cm3) and the average focal volume were computed using this measurement. GSTP and TGF-alpha were demonstrated by irnmunohistochemistry to be expressed in both rat hepatocellular neoplasms and foci. A monoclonal antibody to human TGF-alpha produced 1482
a clear reaction product over hepatocellular carcinomas and adenomas without staining the surrounding unaltered hepatic parenchyma. The frequencies of TGF-alpha-positive tumors are shown in Table I. Most adenomas (82-85%) and all carcinomas (100%) were positively stained using the anti-TGF-alpha antibody. Reaction product was generally expressed uniformly over the hepatocellular elements of the neoplasms. Similar frequencies of neoplasms were positive for expression of GSTP. All hepatocellular carcinomas were positive for GSTP (37/37) and most (24/28) adenomas were positive. The four GSTPnegative adenomas were all classified as amphophilic adenomas (8). One amphophilic adenoma that was tested with antiTGF-alpha antibody was also negative. These results indicated that expression of TGF-alpha is a common feature of GSTP-positive rat hepatocellular neoplasms that were initiated with chemical mutagens and promoted with phenobarbital. In order to determine whether expression of TGF-alpha is also a common feature of GSTP-positive hepatocellular foci, the putative precursors of neoplasms,
Downloaded from http://carcin.oxfordjournals.org/ at New York University on September 11, 2015
B
W.K.Kaufmann, Y.Zhang and D.G.Kaufman
quantitative stereology was used to estimate the numbers of foci with these phenotypic traits (Table H). TGF-alpha-positive foci were seen in only one of seven control livers, whereas all carcinogen-treated livers exhibited positive foci. The frequencies of positive foci when expressed as number per cm or number per cm3 were significantly increased by initiation with BPDE. Due to large variances the average numbers of TGF-alphapositive foci in DMN-OAc-treated rats were not significantly elevated over controls, though the incidence of rats with positive foci was significantly increased by treatment with DMN-OAc. The only control liver that contained three small TGF-alpha foci was estimated by quantitative stereology to contain 170 TGF-alpha-positive foci per cm3. The method of stereologic analysis compensates for the reduced probability of a transection through a small focus, so that small foci when seen in two dimensional sections account for larger numbers in three dimensions (9). Using the stereologic analysis to estimate the yields of foci (as number per cm3), there were 10—30 times more GSTP-positive foci than TGF-alpha-positive foci in treated and control livers.
We have distinguished an additional step in the progression of initiated hepatocellular foci to neoplasms. Quantitative analysis of the frequencies of conversion of hepatocellular foci to neoplasms (6,7) implied that only a subtraction of foci ever develop into neoplasms. In the current sample of carcinogentreated livers, one neoplasm developed for every 2000—20 000 GSTP-positive foci (manuscript in preparation). Only a minor fraction of these GSTP-positive foci expressed TGF-alpha (3 - 7 %). The fact that expression of TGF-alpha was as frequently seen in neoplasms as was expression of GSTP suggests that only the subset of GSTP-positive foci which also express TGF-alpha will progress to neoplasia during promotion with phenobarbital. Thus, acquisition of expression of TGF-alpha appears to be an additional step in the progression of initiated foci to neoplasms.
The majority (65%) of TGF-alpha-positive foci in carcinogentreated livers displayed a heterogeneous distribution of reaction product with anti-TGF-alpha antibody. Typically one margin of the focus, especially one facing onto a blood vessel, was strongly stained while the remainder of the focus displayed a staining intensity near the background (Figure 1A). This feature is reminiscent of a recent demonstration that in adult mice glutamine synthetase and ornithine aminotransferase genes are expressed only in a single-cell-thick rim of hepatocytes surrounding hepatic collecting veins (10). Less frequently there was clear evidence for a TGF-alpha-positive focus within a GSTP-positive focus and the zonal pattern of expression of TGF-alpha was less apparent (Figure 1C). Because the GSTP-positive foci are likely to represent clones of initiated hepatocytes (11), the demonstration of foci-in-foci provides evidence for an additional step in the clonal evolution of these preneoplastic precursors. In contrast to the typical heterogeneous staining for TGF-alpha in foci, staining for GSTP was uniform (Figure IB and D). In transgenic mice that overexpress TGF-alpha in livers, hepatocellular adenomas and carcinomas may develop early and occur with high incidence (12). Expression of this growth factor within a clone of GSTP-positive hepatocytes could provide a growth advantage and favor further progression to neoplasia. The sizes of TGF-alpha-positive foci and GSTP-positive foci were compared as a measure of relative growth capacity (Table II). The average volumes of the TGF-alpha-positive foci seen in carcinogen-treated livers were significantly greater (7- to 8-fold) than the average volumes of all of the GSTP-positive foci. No significant difference could be discerned between the volumes of foci with zonal or focal expression of TGF-alpha. Increased expression of TGF-alpha mRNA has been seen in proliferating hepatocytes in regenerating liver (1) and so the elevated expression seen in the rat hepatocellular neoplasms and foci may simply reflect their increased rates of growth. Moreover, the non-random, zonal pattern of staining for TGF-alpha in focal areas in contact with collecting veins is suggestive of epigenetic modulation of expression in a specific region of these hyperplastic lesions. It is unclear whether the elevated expression seen in foci and neoplasms represented high mol. wt precursor forms or the mature 50 amino acid TGF-alpha (2). Further studies must be done to determine whether growth of hepatocytes within foci and neoplasms is dependent upon their expression of TGF-alpha, as was suggested by the increased sizes of TGF-alpha-positive foci.
1. Mcad.J.E. and Fausto.N. (1989) Transforming growth factor alpha may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc. Nail. Acad. Sci. USA, 86, 1558-1562. 2. Luetteke.N.C, Michalopoulos.G.K., Teixido.J., Gilmore,R., MassagueJ. and Lee,D.C. (1988) Characterization of high molecular weight transforming growth factor alpha produced by rat hepatocellular carcinoma cells. Biochemistry, 27, 6487-6494. 3. Yeh.Y.-C., Tsai J.-F., Chuang.L.-Y., Yeh,H.-W., Tsai,J.-H., Florine.D.L. and Tam.J.P. (1987) Elevation of transforming growth factor alpha and its relationship to the epidermal growth factor and alpha-fetoprotein levels in patients with hepatocellular carcinoma. Cancer Res., 47, 896-901. 4. GrishamJ.W., Tsao.M.S., Lee.D.C. and Earp.H.S. (1990) Sequential changes in epidermal growth factor receptor/ligand function in cultured rat liver epithelial cells transformed chemically in vitro. Palhobiology, 58, 3 — 14. 5. Walker.C, Everitt.J., FreedJ.J., Knudson,A.G. and Whheley.L.O. (1991) Altered expression of transforming growth factor-alpha in hereditary rat renal cell carcinoma. Cancer Res., 51, 2973-2978. 6. Kaufmann.W.K., MacKenzie.S.A. and Kaufman,D.G. (1985) Quantitative relationship between neoplasms and islands of cellular alteration during hepatocarcinogenesis in the male F344 rat. Am. J. PathoL, 119, 171 — 174. 7. Kaufmann.W.K., Rahija.R.J., MacKenzie.S.A. and Kaufman.D.G. (1987) Cell cycle-dependent initiation of hepatocarcinogenesis in rats by ( ± yjr-Std^ydroxy-9r,10/-epoxy-7,8,9,10-tetrahydrobenzc>(a,)pyrene. Cancer Res., 47, 3771-3775. 8. Maronpot,R.R., Montgomery,C.A., Boorman.G.A. and McConnell.E.E. (1986) National Toxicology Program nomenclature for hepatoproliferative lesions in rats. Toxicol. PathoL, 14, 263-273. 9. Pugh.T.D., KingJ.H., Koen.H., Nychka.D., ChoverJ., Wahba.G., He,Y. and Goldfarb.S. (1983) Reliable stereological method for estimating the number of microscopic hepatocellular foci from their transections. Cancer Res., 43, 1261-1268. 10. Kuo.F.C. and DarnelU.E., Jr (1991) Evidence that interaction of hepatocytes with the collecting (hepatic) vein triggers position-specific transcription of the glutamine synthetase and omithine aminotransferase genes in mouse liver. Mol. Cell. Bioi, 11, 6050-6058. 11. Weinberg.W.C, Berkwits.L. and Iannaccone.P.M. (1987) The clonal nature of carcinogen-induced altered foci of gamma-glutamyl transpeptidase expression in rat liver. Carcinogenesis, 8, 565—570. 12. Jhappan.C, Stahle.C, Harkins.R.N., Fausto.N., Smith.G.H. and Merlino.G.T. (1990) TGF-alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of mammary gland and pancreas. Cell, 61, 1137-1146.
Acknowledgements Supported by PHS grant CA42765.
Received on March 3, 1992; revised on May 4, 1992; accepted on May 8, 1992
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References
SHORT COMMUNICATION
Association between expression of transforming growth factor-alpha and progression of hepatocellular foci to neoplasms
William K.Kaufmann, Yingchun Zhang and David G.Kaufman Department of Pathology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA
Transforming growth factor-alpha (TGF-alpha*) is a growth factor for hepatocytes that is produced by proliferating hepatocytes in regenerating livers (1) as well as by a variety of tumor cells including a rat hepatocellular carcinoma line (2). Urinary TGF-alpha has been proposed as a tumor marker for management of patients with hepatocellular carcinoma (3). Analysis of phenotypic alterations that co-segregate with tumorigenesis in a liver epithelial cell culture model of carcinogenesis indicated that expression of TGF-alpha and acquisition of an autocrine growth cycle were positively correlated with tumorigenicity (4). We have used a commercial monoclonal antibody that was raised against human TGF-alpha to test for alterations in expression of TGF-alpha in rat hepatocellular neoplasms. This antibody preparation had been used to demonstrate elevated expression of TGF-alpha in renal carcinomas that
•Abbreviations: TGF-alpha, transforming growth factor-alpha; BPDE, (±)-7r,8»-dihydroxy-9r,10»-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; DMN-OAc, methyl(acetoxymethyl)nitrosamine; GSTP, glutathione-S-transferase (placental isoform). © Oxford University Press
Sections from the right lateral lobe that were free of neoplasms were processed for quantitation of altered hepatocellular foci. Serial paraffin-embedded sections of 5 /im were cut and stained for expression of glutathione-S-transferase (placental isoform) (GSTP) and TGF-alpha. Immunohistochemical staining for GSTP was performed according to the method described in the StrAviGen FASTest Universal Immunostaining Kit purchased from BioGenex Laboratories, San Ramon, CA. Anti-GSTP polyclonal antibody was kindly provided by Dr Ross Cameron, Toronto General Hospital, Toronto, Canada. A mouse monoclonal antibody to human TGF-alpha was obtained from Table I. Incidence of hepatocellular neoplasms that expressed TGF-alpha
Adenomas
DMN-OAc Carcinomas
BPDE Adenomas
Carcinomas
14/17" 82%
18/18 100%
11/13 85%
23/23 100%
"One TGF-alpha-negative adenoma was a GSTP-negative amphophilic adenoma.
1481
Downloaded from http://carcin.oxfordjournals.org/ at New York University on September 11, 2015
Hepatocardnogenesis was initiated in rats with a single dose of either of two chemical mutagens—benzo[a]pyrene diolepoxide I and methyl(acetoxymethyl)nitrosamine—administered 15 h after partial hepatectomy. Hie development of hepatocellular foci and neoplasms was then promoted with dietary phenobarbital given for 45 or 62 weeks. Formalin-fixed tissue specimens that contained hepatic neoplasms and altered hepatocellular foci were screened for expression of the oncodevelopmental marker glutathione-S-transferase (placental form) (GSTP) and transforming growth factoralpha (TGF-alpha) using immunohistochemistry. All (100%) hepatocellular carcinomas expressed both GSTP and TGFalpha, as did most hepatocellular adenomas (>80%). However, quantitative stereologic analysis of treated and control livers revealed that GSTP-positive foci were 10-30 tunes more frequent than TGF-alpha-positive foci. Foci with homogeneous expression of GSTP generally displayed heterogeneous expression of TGF-alpha with reaction product most prominent at their peripheries. Less frequently homogeneous TGF-alpha-positive foci were seen within GSTP-positive foci. The average volumes of those GSTPpositive foci that also expressed TGF-alpha were significantly greater than those of the entire sets of GSTP-positive foci. These results suggest that expression of TGF-alpha may distinguish a subset of GSTP-positive foci that have a growth advantage and increased probability of progression to neoplasia.
develop in a hereditary rat model (5). The current analysis revealed a high frequency of expression of TGF-alpha in hepatocellular neoplasms. However, when foci of hepatocellular alteration were examined, these lesions expressed TGF-alpha less frequendy and those that did usually displayed a heterogeneous staining pattern. The results suggest that acquisition of an autocrine growth cycle involving TGF-alpha may be an uncommon change in hepatocellular foci that heralds an increased probability of progression to neoplasia. Details of treatment of rats with (±)-7r,8/-dihydroxy-9r,10repoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and methyl (acetoxymethyl)nitrosamine (DMN-OAc) were as described previously (6,7). BPDE was purchased from Midwest Research Institute, Kansas City, MO. DMN-OAc was provided by Dr Jerry Rice, Frederick Cancer Research and Development Facility. Phenobarbital was from Sigma Chemical Co., Kansas City, MO. The diet ATN-76A with and without 0.05% phenobarbital was obtained from Zeigler Bros. Inc., Gardners, PA. F344 male rats were obtained from Charles River Breeding Laboratories, Raleigh, NC, at 6 weeks of age. One week after receipt rats were subjected to a two-thirds partial hepatectomy. Fifteen hours later BPDE and DMN-OAc were administered directly into the portal vein at a dosage of 0.02 and 0.1 mmol/kg respectively. Three weeks after treatment with BPDE and DMN-OAc (or after partial hepatectomy for controls), phenobarbital was added to the diet at 0.05%. Rats were fed this diet until 1 week before they were killed, when phenobarbital was withdrawn. At 45 and 62 weeks after initiation, rats were killed by exsanguination when under ether anesthesia. Livers were fixed in 10% buffered formalin, and then sectioned at 1 - 2 mm intervals. Visible tumor masses > 1 mm in diameter were identified and saved for paraffin embedding and histologic diagnosis in H&E-stained sections. Histopathological criteria for diagnosis of hepatocellular adenoma and carcinoma were as suggested by Maronpot et al. (8).
TGF-alpha in rat hepatocarcinogenesis
Table II. Incidences, numbers and volumes of GSTP-positive and TGF-alpha-positive hepatocellular foci Control
Incidence No./cm 2 No./cm 3 Mean volume ( x 10" 3 cm3)
GSTP
TGF-alpha
DMN-OAc GSTP
8/8 3.3 ± 2.4 247 ± 139 0.2 ± 0.1
1/7 0.4 ± l.l c 24 ± 64C 0.1 ± 0.1
5/5 18 ± 4" 487 ± 89" 2.3 ± 1.4"
TGF-alpha 5/5*
2± r 33 ± 31C 16.8 ± 10.8 bc
BPDE GSTP
TGF-alpha
9/9 42 ± 23" 1732 ± 1165" 1.4 ± 0.9"
9/9" 4 ± 2"-c 55 ± 3l"' c 9.5 ± 7.8"-c
Rats were given a single administration of DMN-OAc or BPDE at 15 h after partial hepatectomy followed by dietary exposure to phenobarbital. Controls received a partial hepatectomy and then dietary phenobarbital as for the carcinogen-treated groups. Livers were harvested 45 weeks after partial hepatectomy (controls) or 45—62 weeks after carcinogen treatment and processed for lmmunohistochemical demonstration of TGF-alpha and GSTP. Quantitation of foci as number/cm3 was done by quantitative stereology as described by Pugh el al. (9). 'Significantly different from control (chi-squared test, P < 0.05). Incidence is the number of positive livers divided by the number examined. "Significantly different from control (Student's t-test, P < 0.05). c Significantly different from GSTP (Student's (-test, P < 0.05).
Fig. 1. Expression of TGF-alpha and GSTP in hepatocellular foci. (A) Hepatocellular focus showing heterogeneous expression of TGF-alpha with prominent reaction product at periphery adjacent to collecting veins; (B) serial section containing the focus in (A) stained for GSTP; ( Q focus of more homogeneous expression of TGF-alpha; (D) serial section containing the focus in (C) stained for GSTP. Photomicrographs were made at 400x magnification. All sections were counter-stained with hematoxylin.
Oncogene Science, Manhasset, NY. After incubation of the antibody with paraffin sections for 18 h, visualization of antiTGF-alpha antibody binding was done using the FASTest kit. We reproduced the control observation of Walker ex al. (5) that preincubation of anti-TGF-alpha antibody with human recombinant TGF-alpha abolished immunohistcchemical reaction product over neoplasms and foci, whereas preincubation of the primary antibody with epidermal growth factor did not. Altered hepatocellular foci were quantified according to the method of Pugh et al. (9). Briefly, using a stereomicroscope equipped with a camera lucida, focal outlines were traced onto a digitizing tablet. A computer software program (BIOQUANT System IV, R&M Biometrics, Inc., Nashville, TN) calculated the area of each focus and the radius was determined assuming foci were spheroids. The numerical density of foci (number/cm3) and the average focal volume were computed using this measurement. GSTP and TGF-alpha were demonstrated by irnmunohistochemistry to be expressed in both rat hepatocellular neoplasms and foci. A monoclonal antibody to human TGF-alpha produced 1482
a clear reaction product over hepatocellular carcinomas and adenomas without staining the surrounding unaltered hepatic parenchyma. The frequencies of TGF-alpha-positive tumors are shown in Table I. Most adenomas (82-85%) and all carcinomas (100%) were positively stained using the anti-TGF-alpha antibody. Reaction product was generally expressed uniformly over the hepatocellular elements of the neoplasms. Similar frequencies of neoplasms were positive for expression of GSTP. All hepatocellular carcinomas were positive for GSTP (37/37) and most (24/28) adenomas were positive. The four GSTPnegative adenomas were all classified as amphophilic adenomas (8). One amphophilic adenoma that was tested with antiTGF-alpha antibody was also negative. These results indicated that expression of TGF-alpha is a common feature of GSTP-positive rat hepatocellular neoplasms that were initiated with chemical mutagens and promoted with phenobarbital. In order to determine whether expression of TGF-alpha is also a common feature of GSTP-positive hepatocellular foci, the putative precursors of neoplasms,
Downloaded from http://carcin.oxfordjournals.org/ at New York University on September 11, 2015
B
W.K.Kaufmann, Y.Zhang and D.G.Kaufman
quantitative stereology was used to estimate the numbers of foci with these phenotypic traits (Table H). TGF-alpha-positive foci were seen in only one of seven control livers, whereas all carcinogen-treated livers exhibited positive foci. The frequencies of positive foci when expressed as number per cm or number per cm3 were significantly increased by initiation with BPDE. Due to large variances the average numbers of TGF-alphapositive foci in DMN-OAc-treated rats were not significantly elevated over controls, though the incidence of rats with positive foci was significantly increased by treatment with DMN-OAc. The only control liver that contained three small TGF-alpha foci was estimated by quantitative stereology to contain 170 TGF-alpha-positive foci per cm3. The method of stereologic analysis compensates for the reduced probability of a transection through a small focus, so that small foci when seen in two dimensional sections account for larger numbers in three dimensions (9). Using the stereologic analysis to estimate the yields of foci (as number per cm3), there were 10—30 times more GSTP-positive foci than TGF-alpha-positive foci in treated and control livers.
We have distinguished an additional step in the progression of initiated hepatocellular foci to neoplasms. Quantitative analysis of the frequencies of conversion of hepatocellular foci to neoplasms (6,7) implied that only a subtraction of foci ever develop into neoplasms. In the current sample of carcinogentreated livers, one neoplasm developed for every 2000—20 000 GSTP-positive foci (manuscript in preparation). Only a minor fraction of these GSTP-positive foci expressed TGF-alpha (3 - 7 %). The fact that expression of TGF-alpha was as frequently seen in neoplasms as was expression of GSTP suggests that only the subset of GSTP-positive foci which also express TGF-alpha will progress to neoplasia during promotion with phenobarbital. Thus, acquisition of expression of TGF-alpha appears to be an additional step in the progression of initiated foci to neoplasms.
The majority (65%) of TGF-alpha-positive foci in carcinogentreated livers displayed a heterogeneous distribution of reaction product with anti-TGF-alpha antibody. Typically one margin of the focus, especially one facing onto a blood vessel, was strongly stained while the remainder of the focus displayed a staining intensity near the background (Figure 1A). This feature is reminiscent of a recent demonstration that in adult mice glutamine synthetase and ornithine aminotransferase genes are expressed only in a single-cell-thick rim of hepatocytes surrounding hepatic collecting veins (10). Less frequently there was clear evidence for a TGF-alpha-positive focus within a GSTP-positive focus and the zonal pattern of expression of TGF-alpha was less apparent (Figure 1C). Because the GSTP-positive foci are likely to represent clones of initiated hepatocytes (11), the demonstration of foci-in-foci provides evidence for an additional step in the clonal evolution of these preneoplastic precursors. In contrast to the typical heterogeneous staining for TGF-alpha in foci, staining for GSTP was uniform (Figure IB and D). In transgenic mice that overexpress TGF-alpha in livers, hepatocellular adenomas and carcinomas may develop early and occur with high incidence (12). Expression of this growth factor within a clone of GSTP-positive hepatocytes could provide a growth advantage and favor further progression to neoplasia. The sizes of TGF-alpha-positive foci and GSTP-positive foci were compared as a measure of relative growth capacity (Table II). The average volumes of the TGF-alpha-positive foci seen in carcinogen-treated livers were significantly greater (7- to 8-fold) than the average volumes of all of the GSTP-positive foci. No significant difference could be discerned between the volumes of foci with zonal or focal expression of TGF-alpha. Increased expression of TGF-alpha mRNA has been seen in proliferating hepatocytes in regenerating liver (1) and so the elevated expression seen in the rat hepatocellular neoplasms and foci may simply reflect their increased rates of growth. Moreover, the non-random, zonal pattern of staining for TGF-alpha in focal areas in contact with collecting veins is suggestive of epigenetic modulation of expression in a specific region of these hyperplastic lesions. It is unclear whether the elevated expression seen in foci and neoplasms represented high mol. wt precursor forms or the mature 50 amino acid TGF-alpha (2). Further studies must be done to determine whether growth of hepatocytes within foci and neoplasms is dependent upon their expression of TGF-alpha, as was suggested by the increased sizes of TGF-alpha-positive foci.
1. Mcad.J.E. and Fausto.N. (1989) Transforming growth factor alpha may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc. Nail. Acad. Sci. USA, 86, 1558-1562. 2. Luetteke.N.C, Michalopoulos.G.K., Teixido.J., Gilmore,R., MassagueJ. and Lee,D.C. (1988) Characterization of high molecular weight transforming growth factor alpha produced by rat hepatocellular carcinoma cells. Biochemistry, 27, 6487-6494. 3. Yeh.Y.-C., Tsai J.-F., Chuang.L.-Y., Yeh,H.-W., Tsai,J.-H., Florine.D.L. and Tam.J.P. (1987) Elevation of transforming growth factor alpha and its relationship to the epidermal growth factor and alpha-fetoprotein levels in patients with hepatocellular carcinoma. Cancer Res., 47, 896-901. 4. GrishamJ.W., Tsao.M.S., Lee.D.C. and Earp.H.S. (1990) Sequential changes in epidermal growth factor receptor/ligand function in cultured rat liver epithelial cells transformed chemically in vitro. Palhobiology, 58, 3 — 14. 5. Walker.C, Everitt.J., FreedJ.J., Knudson,A.G. and Whheley.L.O. (1991) Altered expression of transforming growth factor-alpha in hereditary rat renal cell carcinoma. Cancer Res., 51, 2973-2978. 6. Kaufmann.W.K., MacKenzie.S.A. and Kaufman,D.G. (1985) Quantitative relationship between neoplasms and islands of cellular alteration during hepatocarcinogenesis in the male F344 rat. Am. J. PathoL, 119, 171 — 174. 7. Kaufmann.W.K., Rahija.R.J., MacKenzie.S.A. and Kaufman.D.G. (1987) Cell cycle-dependent initiation of hepatocarcinogenesis in rats by ( ± yjr-Std^ydroxy-9r,10/-epoxy-7,8,9,10-tetrahydrobenzc>(a,)pyrene. Cancer Res., 47, 3771-3775. 8. Maronpot,R.R., Montgomery,C.A., Boorman.G.A. and McConnell.E.E. (1986) National Toxicology Program nomenclature for hepatoproliferative lesions in rats. Toxicol. PathoL, 14, 263-273. 9. Pugh.T.D., KingJ.H., Koen.H., Nychka.D., ChoverJ., Wahba.G., He,Y. and Goldfarb.S. (1983) Reliable stereological method for estimating the number of microscopic hepatocellular foci from their transections. Cancer Res., 43, 1261-1268. 10. Kuo.F.C. and DarnelU.E., Jr (1991) Evidence that interaction of hepatocytes with the collecting (hepatic) vein triggers position-specific transcription of the glutamine synthetase and omithine aminotransferase genes in mouse liver. Mol. Cell. Bioi, 11, 6050-6058. 11. Weinberg.W.C, Berkwits.L. and Iannaccone.P.M. (1987) The clonal nature of carcinogen-induced altered foci of gamma-glutamyl transpeptidase expression in rat liver. Carcinogenesis, 8, 565—570. 12. Jhappan.C, Stahle.C, Harkins.R.N., Fausto.N., Smith.G.H. and Merlino.G.T. (1990) TGF-alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of mammary gland and pancreas. Cell, 61, 1137-1146.
Acknowledgements Supported by PHS grant CA42765.
Received on March 3, 1992; revised on May 4, 1992; accepted on May 8, 1992
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