The little mutation suppresses DEN-induced hepatocarcinogenesis in mice and abrogates genetic and hormonal modulation of susceptibility

James M. Bugni,1, Therese M. Poole,2 and Norman R. Drinkwater,3

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Avenue, Madison, WI 53706, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In mice, the sex difference in susceptibility to hepatocarcinogenesis results from the tumor promoting activity of testosterone and from the inhibition of tumor promotion by ovarian hormones. We investigated the role of growth hormone in the sex-dependent regulation of susceptibility, because sex hormones are known to regulate the temporal pattern of growth hormone secretion and subsequent sex differences in liver gene expression. We found that in both males and females, wild-type mice developed significantly more tumors than growth hormone-deficient, C57BL/6J-lit/lit (B6-lit/lit) mutant mice following perinatal treatment with the carcinogen N,N-diethylnitrosamine (DEN). B6 wild-type males developed 36–59-fold more liver tumors per animal than age matched B6-lit/lit males and wild-type females developed 11-fold more tumors than B6-lit/lit females. We bred the little mutation onto the more susceptible C57BR/cdJ (BR) and C3H/HeJ (C3H) strains to assess the effect of growth hormone deficiency on hepatocarcinogenesis on additional genetic backgrounds. Growth hormone deficiency suppressed liver tumor development to <1% in males of each strain and in BR strain females. In B6 and C3H females, growth hormone deficiency caused 2–4-fold reductions in the volume fraction of the liver occupied by preneoplastic lesions. Furthermore, in contrast to wild-type strains, neither gonadectomy nor strain background significantly affected susceptibility in lit/lit mice, as mean liver tumor multiplicities ranged from 0 to 0.24 ± 0.44 and the volume fraction of preneoplastic lesions ranged from 0.21 ± 0.22 to 0.61 ± 1.9%. These results demonstrate that both strain and sex hormonal effects on susceptibility to liver carcinogenesis are dependent on wild-type levels of growth hormone.

Abbreviations: B6, C57BL/6J; BR, C57BR/cdJ; BrdU, bromodeoxyuridine; C3H, C3H/HeJ; DEN, N,N-diethylnitrosamine; lit, little; Tfm, Testicular feminization.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Male mice are more susceptible than females to spontaneous liver cancer (1,2) and to the induction of liver tumors by a wide variety of carcinogens (39). Endocrine ablation studies have demonstrated that the sex difference in susceptibility results from the opposing effects of male and female sex hormones, with testosterone enhancing (1,3,5,1014) and ovarian hormones inhibiting (3,1014) liver tumor development. Following perinatal treatment with N,N-diethylnitrosamine (DEN) (DEN preweanling model), ovariectomy causes an 8-fold increase in liver tumor multiplicity in C57BL/6J (B6) and C3H/HeJ (C3H) mice, and a modest increase in C57BR/cdJ (BR) mice, while castration in all three strains causes a 3–14-fold reduction in the number of liver tumors relative to intact mice (3). Analyses of microscopic lesions and the kinetics of tumor development have demonstrated that sex hormones affect the growth of preneoplastic foci (3,10,1316).

Although it has been well established in mice that gonadal sex hormones have dramatic effects on susceptibility to hepatocarcinogenesis, the molecular and physiological mechanisms mediating these effects are not well understood. Studies using Testicular feminization (Tfm) mutant mice, which have a germline, X-linked mutation in the androgen receptor gene (17), showed that tumor promotion by testosterone is dependent on a functional androgen receptor, but must be mediated by a secondary, secreted factor (15). Although the mechanism by which ovarian hormones inhibit hepatocarcinogenesis is less clear, it is likely to be mediated by estrogens (18,19).

Several observations led us to test the hypothesis that pituitary growth hormone mediates the sex difference in susceptibility to hepatocarcinogenesis. In both mice and rats, males and females differ markedly in the temporal pattern of growth hormone secretion and these different secretory patterns are responsible for a variety of sex differences in liver gene expression (2023). Moreover, pulsatile infusion of growth hormone is sufficient to masculinize, and continuous infusion is sufficient to feminize, the expression of MUP, prolactin receptor and testosterone 16-{alpha} hydroxylases in mouse liver (23,24).

The hypothesis that growth hormone promotes liver carcinogenesis also followed from early studies in the rat that showed a reduction in the incidence of induced hepatomas following hypophysectomy (2530). These studies further showed that tumor development was enhanced in hypophysectomized rats when they were treated with growth hormone, albeit not to the same levels as intact rats (27,28). However, administration of other pituitary hormones (gonadotropin and TSH) also had minor restorative effects, and adrenocorticotropic hormone had the same effect as growth hormone. These results are difficult to interpret with respect to the effect of growth hormone because of impurities in the replaced hormones and because of the pleiotropic effects of the pituitary on the hormonal status and metabolism in the animals. In the Solt–Farber model of rat hepatocarcinogenesis, continuous infusion of growth hormone, explantation of pituitary grafts and castration act similarly in males to feminize the liver with respect to metabolism and preneoplastic focus development (3134). In mice, the effect of growth hormone overexpression on hepatocarcinogenesis is evident in transgenic animals (3537). Mice expressing ovine growth hormone at serum levels ~4000-fold higher than normal develop chronic hepatitis and eventually spontaneous hepatocellular carcinomas (35,36).

The little (lit) mutation, which arose spontaneously in the B6 strain (38), is a point mutation in the gene for the growth hormone releasing hormone receptor (Ghrhrlit) (39,40). In mutant mice, somatotroph cells in the pituitary do not respond to releasing hormone from the hypothalamus, and thus, serum levels of growth hormone in B6-lit/lit adults are ~5% of the normal level (41,42). These mice have significantly reduced body weights and bone lengths compared with phenotypically normal, heterozygous mice, but food consumption per body weight is similar (43). We chose to study B6-lit/lit mice to determine the effects of growth hormone on liver carcinogenesis, because unlike the other known mutant mice with growth hormone deficiencies, specifically the Ames and Snell Dwarfs, B6-lit/lit mice have normal serum levels of other pituitary hormones (42,44,45).

We first compared B6-lit/lit to B6 wild-type mice in order to determine the effects of growth hormone on DEN-induced hepatocarcinogenesis. Growth hormone deficiency dramatically suppressed liver tumor development in both males and females on a B6 background. To assess the generality of growth hormone's effects on liver tumor development, in subsequent studies we analyzed DEN-induced hepatocarcinogenesis in lit/lit and wild-type mice on two additional genetic backgrounds that are highly susceptible to liver tumor induction, C3H and BR (3). Genetic background can have a dramatic effect on susceptibility to hepatocarcinogenesis and several genetic loci have been mapped that control susceptibility (4650). For the strains used in this study, the Hepatocarcinogen susceptibility 7 (Hcs7) locus was mapped in crosses of B6 and C3H mice (49) and the Hepatocarcinogenesis in females 1 and 2 (Hcf1 and Hcf2) loci were mapped in crosses of B6 to BR mice (48). Of these three strains, in males, C3H mice are most susceptible, while in females, BR mice are most susceptible. This sex difference in relative susceptibilities is due in part to the resistance of BR females to the inhibitory effects of ovarian hormones (3).

Finally, we performed gonadectomies on mutant and wild type mice to determine whether the sex-dependent effects on susceptibility to hepatocarcinogenesis require growth hormone. We found that growth hormone deficiency suppressed liver tumor development to the same absolute level regardless of the strain background or the hormonal environment.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animal husbandry
BR, C3H, B6 and B6-lit/lit mice were obtained from the Jackson Laboratory (Bar Harbour, ME) and bred in our laboratory. Mice were raised on acidified tap water (pH 2.65) and were fed Breeder Blox ad libitum (11% fat; Purina, St Louis, MO) (Tables I and IVGoGo). On this diet, B6-lit/lit mice were maintained as a segregating inbred by lit/lit x lit/+ crosses, because homozygous males had reduced fertility. Alternatively, mice were fed NIH31-6% (low) fat diet ad libitum (Harlan-Teklad, Madison, WI) (Tables II and IIIGoGo), to allow breeding of lit/litxlit/lit mice and to reduce the incidence of excoriative dermatitis in B6 mice (Jackson Laboratory, http://jaxmice.jax.org/html/archives/c57update.shtml). Mice were assigned to experimental groups by visual inspection of their size at the time of weaning, and the inferred genotypes were confirmed after necropsy by the PCR–RFLP assay described below.


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Table I. Liver tumor development in B6 and B6-lit/lit male and female micea
 

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Table IV. Effects of gonadectomy on the early development of hepatic preneoplastic foci in three wild-type strains of micea
 

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Table II. Effect of growth hormone deficiency and of gonadectomy on hepatocarcinogenesis in three strains of micea
 

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Table III. Effect of gonadectomy on the development of preneoplastic foci in three strains of lit/lit micea
 
Generation of congenic C3H-lit/lit and BR-lit/lit mice
C3HxB6-lit/lit and BRxB6-lit/lit matings were established and F1 males were backcrossed to C3 or BR females, respectively. Offspring were genotyped for the little mutation and heterozygous males were selected for subsequent backcrosses. Fifth generation (4th backcross; N5) congenic animals were bred in three independent lines for the BR recipients and four independent lines for C3H recipients. These fifth generation lit/+ mice were intercrossed and offspring of the three possible genotypes +/+, lit/+ and lit/lit from two or three generations after intercrossing were used in tumorigenesis studies.

A single locus on chromosome 1 (Hcs7) is responsible for most of the difference in hepatocarcinogen susceptibility between C3H and B6 and two loci on chromosomes 17 and 1 account for virtually all of the difference in susceptibility between BR and B6 (48,49). These susceptibility loci are unlinked to the little mutation on chromosome 6 (38). We genotyped our N5 congenic mice to ensure that the chromosomal regions containing these susceptibility loci were homozygous for the BR or C3H genotypes. N5 congenics would be expected to carry ~3% residual donor strain genetic background at loci unlinked to the selected genes. Therefore, roughly equal numbers of mice from the independent lines were used in each experimental group in order to minimize the effects of residual heterozygosity on susceptibility to hepatocarcinogenesis.

Gonadectomy
Four-week-old (Tables II and IIIGoGo) or 8-week-old (Table IVGo) mice were anaesthetized with avertin (0.25 µg/g body wt; Sigma, St Louis, MO) and a single longitudinal incision was made through the ventral skin for castrations or through the dorsal skin for ovariectomies. For castrations, the vas deferens and associated vessels were cauterized transperitoneally and the testes and epididymis were excised. For ovariectomies, two small incisions were made in the dorsal peritoneum lateral to the spine. The Fallopian tubes and associated vessels were cauterized and the ovaries excised.

Carcinogenesis studies
Twelve-day-old mice were injected intraperitoneally (i.p.) with DEN at doses of 0.1 (Table IGo) or 0.05 (Tables II–IVGoGoGo) µmol/g body wt in sterile tricaprylin (Sigma). Mice were killed at 24, 32 or 50 weeks of age and tumors >1 mm in diameter on the surface of the liver were enumerated. The Wilcoxon rank sum test was used to determine the statistical significance of differences in tumor multiplicity between two groups and the Kruskal–Wallis test was used to test for differences among more than two groups. When comparing groups with low tumor multiplicities, we also used Fisher's exact test to determine the significance of differences in tumor incidence. To account for the multiple comparisons made within an experiment, all P values were adjusted to experiment-wise values using the sequential Dunn–Sidak method (51). For the studies on the effect of growth hormone deficiency and gonadectomy on hepatocarcinogenesis and the development of preneoplastic foci (Tables I and IIGoGo), we made six and 50 tests, respectively.

Analysis of microscopic lesions
The majority of microscopic preneoplastic lesions that develop in wild-type (52,53) and lit/lit (J.Bugni and N.Drinkwater, unpublished data) mice are glucose-6-phosphatase (G6Pase)-deficient, basophilic and overexpress Bcl-2. In the present study we quantified basophilic foci (Table IIIGo) or G6Pase-deficient foci (Table IVGo). After enumerating tumors, ~5 mm sections were cut from the left lateral and left and right portions of the median lobe and fixed in 10% phosphate buffered formalin. Sections (4 µm) were cut and stained with hematoxylin and eosin (H&E). Sixteen sections from lit/lit mice and three to five sections from each group of wild-type mice were selected at random for histopathological diagnosis. Alternatively, livers were frozen on dry ice for G6Pase staining by a modification of the method of Wachstein and Meisel (54). Frozen sections were fixed briefly in 0.5% glutaraldehyde and were incubated at 37°C for 45 min in 2% lead nitrate and 0.5 mg/ml glucose-6-phosphate in 0.1 M Tris–maleate (pH 7.2). Slides were then incubated in 7.5% ammonium sulfide and fixed in 5% glutaraldehyde and 5% acetic acid. Sections were counterstained with hematoxylin, rinsed in an ethanol series (50, 70, 95 and 100%) for 20 min and rinsed four times with xylene prior to mounting.

Basophilic or G6Pase-deficient foci were enumerated and their diameters measured with a calibrated ocular micrometer. Sections were scanned and the total section areas were measured using Scion image analysis software (Scion Corp; Frederick, MD). The average area sampled was 0.95 ± 0.32 cm2 per animal. Mean focal diameters were determined as described previously using the number of focus transections per section, their diameters and the area of section (55). Mean values were calculated from pooled data for each experimental group and individual measurements were used to determine standard deviations and statistical significance using the Wilcoxon rank sum and Kruskal–Wallis tests. P values were adjusted to experiment-wise values using the sequential Dunn–Sidak method and for microscopic studies shown in Tables III and IVGoGo, 18 and 23 tests were performed, respectively.

PCR–RFLP genotyping for the little mutation
Genotyping of the little mutation was performed as described previously (56) with the following modifications. DNA was purified from mouse tail clips and a 315 base pair (bp) fragment of the growth hormone releasing hormone receptor (Ghrhr) gene was amplified using the primers 5'-ACTGGGTCACCTCCACCTAGAATG-3' and 5'-CCTGTGTCTGAGCCGAAGTGAGAG-3' for 25 cycles. PCR products were digested with the FokI restriction endonuclease and electrophoresed on 9% polyacrylamide gels. B6-lit/lit mice carry an A->G transition in the Ghrhr gene that abolishes a FokI restriction site. Homozygous lit/lit mice were characterized by the presence of a 96 bp diagnostic band and a 76 bp diagnostic band characterized wild-type mice. Both bands are present in the heterozygote.

Hepatocyte incorporation of BrdU
To assess bromodeoxyuridine (BrdU) incorporation, three or four 12-day-old mice of each sex and genotype (B6 and B6-lit/lit) were injected i.p. with 0.4 mg/g body wt BrdU (20 mg/ml in PBS; Sigma). Mice were injected between 9 and 10 p.m. and 12 h later livers were removed, rinsed with 0.9% saline and fixed in 10% phosphate buffered formalin.

Immunohistochemical detection of BrdU was performed by standard procedures (57). Briefly, endogenous peroxidase was blocked with 3% hydrogen peroxide. Sections were digested with protease and subjected to unincorporated antigen retrieval with 5% urea. Sections were blocked with 0.05 mg/ml avidin, 0.05 mg/ml biotin and BLOTTO. Slides were incubated with a 1:50 dilution of {alpha}-BrdU (M0744; Dako, Carpinteria, CA) at 4°C for 12 h. A 1:700 dilution of biotinylated goat anti-mouse IgG (Zymed, San Francisco, CA) was applied for 2 h at room temperature and slides were incubated with streptavidin–horseradish peroxidase (1:250 in PBS; Zymed) for 30 min at room temperature. The red color was developed with AEC reagent (Zymed) according to the manufacturer's recommendations and sections were counterstained briefly with hematoxylin.

To determine labeling indices, five images for each section (one section/liver) were taken at 213x magnification with an Olympus SZH zoom camera. The area/image was 0.265 mm2 and the total number of nuclei scanned was >24 000 for each group. Images were analyzed using Scion image analysis software (http://scioncorp.com) and Nuclear Labeling Index Analysis software (H.C.Pitot, University of Wisconsin, Madison, WI). Labeled nuclei were selected manually and both total and labeled nuclei were counted automatically.

DEN metabolism assays
Four B6 (three male and one female) and three B6-lit/lit (two male and one female) livers from 12-day-old mice were assayed individually for metabolism of DEN, by quantifying the amount of liberated acetaldehyde. At this age, there is no sex difference in mice in metabolism of DEN (58). Microsomes were prepared by ultracentrifugation and quantification of liberated acetaldehyde was performed using the method of Stotz (59,60) with the following modification: after addition of 5% CuSO4, reactions were clarified by brief centrifugation at 2000 g. Standards were prepared in parallel with each set of reactions by supplementing reaction components (including heat-inactivated microsomes) with known amounts of acetaldehyde.

Experimental design
First, we tested the effect of growth hormone deficiency on hepatocarcinogenesis by comparing tumor multiplicities in B6 wild-type and B6-lit/lit mice. Second, we analyzed the effects of growth hormone in multiple strains by comparing tumor multiplicities and the volume fraction of preneoplastic foci in wild-type and lit/lit B6, BR and C3H mice. In the latter case, mice were gonadectomized or left intact to determine the effect of sex hormones on tumor development in the presence or absence of growth hormone. In both cases, males were killed at 32 or 50 weeks of age and females were killed at 50 weeks of age. Finally, we tested the effect of gonadectomy on the early development of tumors and microscopic foci in wild-type B6, BR and C3H mice that were killed at 24 or 32 weeks of age.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Tumor development in B6 and B6-lit/lit mice
On comparing B6 to B6-lit/lit mice, B6 males at 32 and 50 weeks of age had mean liver tumor multiplicities of 5.9 ± 7.6 and 36 ± 22 and B6-lit/lit males had mean tumor multiplicities of only 0.10 ± 0.31 and 1.0 ± 1.8, respectively (Table IGo). Thus, at 32 and 50 weeks of age B6 males developed 59- and 36-fold more tumors than B6-lit/lit males (P < 10–8). At 50 weeks of age, B6 and B6-lit/lit females exhibited an 11-fold difference in susceptibility with mean tumor multiplicities of 4.2 ± 4.6 and 0.40 ± 1.1, respectively (P < 10–6). The difference in tumor multiplicity between B6 males and B6 females at 50 weeks was 8.6-fold (P < 10–12 ), while B6-lit/lit males and females had only a 2.5-fold difference in tumor multiplicity (P = 0.10) and a 2-fold difference in incidence (P = 0.16).

To determine whether the effect of growth hormone deficiency on tumor multiplicity could be explained by differences in activation of the carcinogen, we measured DEN metabolism by the production of acetaldehyde in microsomal fractions from livers of 12-day-old mice. We found the activities for B6 and B6-lit/lit mice to be nearly identical at 4.3 ± 0.9 and 4.3 ± 0.2 nmol acetaldehyde/mg/min, respectively. Because the carcinogenic potential of DEN depends on proliferation in the liver at the time of treatment, we measured hepatocyte proliferation by BrdU incorporation in 12-day-old B6 and B6-lit/lit mice. B6 males and females had BrdU labeling indices of 2.4 ± 0.3 and 2.6 ± 0.2%, and B6-lit/lit males and females were not significantly lower than wild-type mice, with labeling indices of 2.1 ± 0.5 and 2.2 ± 0.5%, respectively (P > 0.2; Wilcoxon rank sum).

Tumor development in inbred and congenic mice
We determined the effects of growth hormone deficiency on three genetic backgrounds (B6, C3H and BR) (Tables II and IIIGoGo). For our N5 congenic BR and C3H strains, we examined tumor development in +/+, lit/+ and lit/lit mice. There were no differences in tumor multiplicity between lit/+ mice and +/+ mice or among the sublines for each congenic strain. Mean tumor multiplicities in lit/+ and +/+ mice differed by <20% (P > 0.4 ). These results were expected as no biological differences have been found between lit/+ and +/+ mice (43,61) and for these reasons the results from lit/+ and +/+ groups were combined. In the discussion below, normal mice from congenic lines will be designated `BR-+/' or `C3H-+/', and the terms `BR' and `C3H' will be reserved for the pure inbred strains.

BR-+/ and C3H-+/ males had tumor multiplicities comparable with BR and C3H males, respectively, and both groups had significantly more tumors than B6 mice (Table IIGo). At 32 weeks of age BR-+/ and C3H-+/ developed 6.6- and 54-fold more tumors per animal, respectively, than B6 males, which had only 0.59 ± 0.91 tumors per animal (P < 10–4). At 50 weeks of age, there were no significant differences between BR-+/ and C3H-+/ males or between BR and C3H males (P > 0.5), but they were all 2.6–3.7-fold more sensitive than B6 males (P < 10–4).

In females, BR and BR-+/ mice had similar tumor responses that were significantly greater than those for B6 and C3H females. BR-+/ females developed 11 ± 11 tumors, BR females developed 9.1 ± 7.8 tumors; whereas B6, C3H-+/ and C3H inbred females developed <1 tumor per animal. Thus, carrying the congenics to N5 was sufficient to increase tumor responses significantly above the response in B6 mice and to levels comparable with the susceptible recipient strains in both males and females.

Effects of growth hormone deficiency
Growth hormone deficiency suppressed liver tumor development in both sexes on each genetic background and the effect in males was evident at 32 weeks of age (Table IIGo). BR-+/ and C3H-+/ males had mean tumor multiplicities of 3.9 ± 3.8 and 32 ± 32, respectively, while no tumors developed in 21 BR-lit/lit or 21 C3H-lit/lit mice (P < 10–5). One of 16 B6-lit/lit males developed a single tumor, a 9.8-fold lower tumor multiplicity and 6.3-fold lower tumor incidence than in B6 males. However, because of the low tumor response in B6 mice at this age the effect was not significant (P = 0.35). The effects of growth hormone deficiency were more dramatic in 50-week-old male mice. At this age, B6-lit/lit, BR-lit/lit and C3H-lit/lit mice had mean tumor multiplicities of only 0.03 ± 0.19, 0.16 ± 0.50 and 0.16 ± 0.50, which were >1% of the tumor multiplicities of B6, BR-+/ and C3H-+/ males (P < 10–6) (Table IIGo).

In females, B6 mice had a mean liver tumor multiplicity of 0.07 ± 0.27 and B6-lit/lit mice had a tumor multiplicity of 0.04 ± 0.20. In contrast to our first experiment, growth hormone deficiency did not have a significant effect on either tumor multiplicity or incidence in B6 females (P = 1.0). There was a significant effect on the C3H background (P < 0.005, for tumor multiplicity and incidence), as C3H-+/ females had a mean tumor multiplicity of 0.79 ± 2.0 and an incidence of 0.45, and 34 C3H-lit/lit females did not develop any tumors. The effect of growth hormone deficiency was most dramatic in the BR strain, with BR-+/ females developing 11 ± 11 tumors per mouse and no tumors developing in 17 BR-lit/lit females (P < 10–7). Furthermore, there were no significant differences in tumor multiplicity among intact lit/lit mice of all three strains, for either male or female mice (P = 1.0; Kruskal–Wallis).

Effects of gonadectomy
We also tested the effect of gonadectomy in inbred and lit/lit mice on all three strain backgrounds (Table IIGo). By 50 weeks of age, castration of B6, C3H and BR males caused 33-, 5.9- and 2.2-fold reductions, respectively, in tumor multiplicity relative to intact males (P < 10–3). In contrast, tumor multiplicities in 50-week-old lit/lit intact males ranged from 0.03 ± 0.19 to 0.16 ± 0.50 and castration had no effect on susceptibility (P = 1.0).

Ovariectomy has been reported to increase susceptibility in B6 and C3H but not BR mice (3). In the present study, ovariectomy of B6 and C3H mice caused 7.7- and 6.2-fold increases in tumor multiplicity and 5.7- and 3.8-fold increases in tumor incidence, respectively. However, because of the low overall tumor responses in females, the effects were of marginal significance (B6 tumor multiplicity, P = 0.10 and incidence, P = 0.16; C3H tumor multiplicity, P = 0.08 and incidence, P = 0.17). Also, the ~2-fold effect of ovariectomy in BR females was not significant (P = 0.37). Similar to castration, ovariectomy of B6-lit/lit and BR-lit/lit mice had no effect on tumor multiplicity or incidence (P > 0.9). However, 34 intact C3H-lit/lit female mice developed no tumors, while ovariectomized mice had a mean tumor multiplicity of 0.24 ± 0.44. These differences were marginally significant (tumor multiplicity, P = 0.09; incidence, P = 0.14).

Analysis of microscopic foci
For groups in which few tumors developed, we quantified microscopic basophilic foci in order to increase the sensitivity of our analysis. The groups we analyzed included all lit/lit animals, as well as B6 and C3H females (Table IIIGo). We also analyzed microscopic foci in wild-type mice killed at 24 and 32 weeks of age (Table IVGo). Tables III and IVGoGo show the number of foci per cubic centimeter and the percent volume fractions of the liver occupied by preneoplastic lesions. The most robust measure of susceptibility derived from analysis of microscopic lesions is the volume fraction, because it is determined by both the number of preneoplastic foci and their sizes. Furthermore, in contrast to focal diameters and foci per cubic centimeter, the volume fraction can be measured directly, with less sampling error, by measuring the area fraction for each section (55).

There was no difference in tumor multiplicity or incidence between B6-lit/lit and B6 wild-type females (Table IIGo). However, the volume fraction of foci in B6 females was 1.8-fold larger than the volume fraction in B6-lit/lit females (P = 0.02) and the volume fraction in C3H females was 3.8-fold larger than the volume fraction in C3H-lit/lit females (P = 1.4x10-5 ) (Table IIIGo).

At 50 weeks of age, foci in intact lit/lit males constituted from 0.37 ± 0.30 to 0.44 ± 0.26% of the liver volume, a result unaffected by castration (P > 0.94; Table IIIGo). In wild-type mice, however, the inhibitory effects of castration are prominent at early stages of carcinogenesis. At 24 weeks of age, foci in B6 and BR intact males comprised 0.42 ± 0.57 and 7.5 ± 4.8% of the liver volume, whereas foci in castrated males occupied 14- and 19-fold smaller volume fractions, respectively (P < 0.02) (Table IVGo). Castration did not have a significant effect on focus volume fraction in C3H males at 24 weeks of age, but it reduced tumor multiplicity in this strain by 7.1-fold (P = 0.02), as intact males developed 15 ± 21 tumors per animal and castrated males developed 2.1 ± 2.8 tumors per animal.

Similar to castration, ovariectomy had little effect on focus volume fraction in lit/lit mice (Table IIIGo). At 50 weeks of age, intact lit/lit females had percent volume fractions ranging from 0.21 ± 0.22 to 0.61 ± 1.9. Ovariectomy caused a marginally significant, 2.6-fold increase in volume fraction in BR-lit/lit mice (P = 0.09) but did not increase the volume fraction in B6-lit/lit or C3H-lit/lit females (P > 0.32).

In wild-type mice at 24 and 32 weeks of age, foci in ovariectomized females generally occupied larger volume fractions of the liver than in intact mice, but the only significant effects were in the C3H strain (Table IVGo). C3H females, at 24 and 32 weeks of age, had volume fractions of 0.07 ± 0.07 and 0.46 ± 0.33 and ovariectomy caused 4.8- and 16-fold increases, respectively (P < 0.03).

The only marginally significant sex difference in focus volume fraction in lit/lit mice was in the BR strain, in which males had foci occupying a 2.1-fold larger volume fraction than females (P = 0.07) (Table IIIGo). B6-lit/lit and C3H-lit/lit females actually had slightly higher mean volume fractions of foci than males, but the differences were not significant (P > 0.80). In contrast to lit/lit mice, foci in 24- and 32-week-old wild-type males occupied consistently larger volume fractions than in females (Table IVGo). In B6 mice the difference at 24 weeks of age was only 1.3-fold (P = 0.09), but all other differences in volume fraction between males and females were >9-fold (P < 0.01).

There were no strain differences in the volume fractions of hepatic foci among intact lit/lit males or females (P > 0.93; Kruskal–Wallis) (Table IIIGo), but in wild-type mice strain differences were evident in both males and females at early stages (Table IVGo). At 24 weeks of age BR and C3H males, although not significantly different from one another, had 18–29-fold larger focus volume fractions than B6 males (P < 0.004). In females, there were no significant strain differences at 24 weeks of age. At 32 weeks of age, BR females had a 5.9–9.6-fold larger mean volume fraction of foci than B6 and C3H females, but, because of the variation within the groups, the differences were not significant (P > 0.18). However, at this age, BR females developed significantly more tumors than B6 or C3H. Twelve BR females had a tumor multiplicity of 0.50 ± 0.78 and no tumors developed in 13 B6 or 13 C3H females (P = 0.02, for both comparisons).

Of all the lit/lit groups analyzed, the smallest focus volume fractions measured were in the mice killed at 32 weeks of age, demonstrating that foci in lit/lit mice are growing between 32 and 50 weeks of age (Table IIIGo). The difference in volume fraction between B6-lit/lit males at 32 and 50 weeks of age was 1.9-fold (P = 0.77), but the differences in volume fraction between 32- and 50-week-old C3H-lit/lit and BR-lit/lit males were 6.9- and 5.5-fold, respectively (P < 10–3).

Histopathology
Histological analysis of sections through the liver revealed that most lesions in lit/lit mice were small, basophilic preneoplastic foci. Fewer than 40% of lit/lit mice developed microscopic adenomas and their occurrence was independent of strain background and sex. In contrast, all of the wild-type mice examined (50-week-old females, and 32- and 50-week-old males) developed hepatocellular adenomas and these lesions were most abundant in 50-week-old males. In both wild-type and lit/lit mice, adenomas were predominantly type A and ~15% had areas of type B atypia (62).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The magnitude of the effect of wild-type levels of growth hormone on susceptibility to hepatocarcinogenesis is greater than the effect of any other known genetic or hormonal regulator of susceptibility in the DEN preweanling model (3,4). In our first experiment, we showed that growth hormone deficiency dramatically suppressed tumor development in B6 mice, with a greater effect in males than in females. We bred the little mutation onto the more susceptible BR and C3H strain backgrounds for two reasons. First, we sought to determine whether wild-type levels of growth hormone enhance liver tumor development independently of the background strain. Second, we anticipated that a greater number of tumors would develop in lit/lit mice on these sensitive backgrounds, allowing us to better determine the effects of sex hormones in the absence of growth hormone.

Our results clearly demonstrate that on all three strain backgrounds, the little mutation dramatically suppresses liver tumor development. In the second experiment, males with wild-type levels of growth hormone were over 100-fold more sensitive than growth hormone-deficient mice. Tumorigenic responses were on average lower in the second experiment than in the first and the difference was likely due to a combination of the 2-fold lower dose of DEN used and the lower fat content of the diet. For this reason, in females, the only dramatic effect of growth hormone on tumor multiplicity was in the BR strain, but significant effects on the volume fraction of preneoplastic lesions were also observed in B6 and C3H females. The effect of the little mutation on the BR and C3H strain backgrounds demonstrates that the genetic factors that enhance susceptibility in these strains do not overcome the suppressive effects of growth hormone deficiency.

We were surprised by the result that growth hormone deficiency suppressed tumor development to the same absolute level in all strains, as we could not detect significant differences among strains of lit/lit mice in mean tumor multiplicity, tumor incidence or in the volume fraction of preneoplastic lesions. Although very few tumors developed in lit/lit mice, tumor development was not suppressed to a level at which effects of genetic background should go undetected. C3H and BR mice have been shown to develop more preneoplastic foci than B6 mice and in both strains the greater susceptibility relative to B6 mice is in part due to a higher growth rate for hepatic foci (3,55,63). In wild-type mice, we measured significant effects of genetic background on the volume fraction of preneoplastic lesions at 24 and 32 weeks of age. The preneoplastic lesions in lit/lit mice of all three genetic backgrounds at 50 weeks of age are comparable in size and volume fraction to those in wild-type B6 mice at 24 weeks of age. Thus, our failure to detect strain differences among lit/lit mice demonstrates that the major susceptibility factors in C3H and BR mice have either no effect or a severely attenuated effect in the absence of growth hormone.

These strain-dependent susceptibility factors, however, do not act by controlling growth hormone levels. Although C3H mice have been reported to have higher serum levels of growth hormone than B6 mice (64), strain differences in growth hormone secretion cannot be responsible for the majority of the difference in susceptibility to hepatocarcinogenesis in the wild-type strains used in this study. In C3H<-->B6 and BR<-->B6 chimeric mice that were treated with a carcinogen, most hepatocellular neoplasms that developed were derived from the susceptible C3H or BR donor (65,66) (T.Chiaverotti and N.Drinkwater, in preparation). Thus, the major genetic loci controlling susceptibility in these strains act at the level of the target hepatocyte and do not act by controlling growth hormone levels.

Studies using Tfm mutant mice showed that tumor promotion by testosterone must be mediated by a secreted factor (15) and growth hormone is an attractive candidate because it is known to mediate testosterone-dependent sexual dimorphism in the liver (2224). As expected from previous experiments (3), we showed that castration dramatically reduces tumor multiplicity and the volume fraction of preneoplastic lesions in wild-type mice, but gonadectomy does not have a significant effect on susceptibility in lit/lit mice. These results do not prove, but are consistent with, the model that testosterone promotes hepatocarcinogenesis by giving rise to a masculine growth hormone secretory pattern. Another result that supports this hypothesis is the observation that growth hormone has a greater effect on susceptibility in males than in females. In this model, one would expect growth hormone deficiency to have a greater effect in males by nullifying the tumor promoting effect of testosterone.

The results of ovariectomy in lit/lit animals are more difficult to interpret. There were minor effects on tumor multiplicity in C3H-lit/lit females and on focus volume fraction in BR-lit/lit females. Ovariectomy in wild-type females generally increased the volume fraction at 24 and 32 weeks of age, although the only significant effects were the 4–16-fold increases in C3H females. Thus, ovariectomy appears to have a greater effect in wild-type mice than in lit/lit mice. Studies in rats have shown that ovariectomy has little effect on growth hormone levels (67). These results, together with our observations that ovariectomy may have a small effect in lit/lit mice, suggest that ovarian hormones probably do not suppress liver tumor development by modifying growth hormone levels.

The only unambiguous modifier of preneoplastic lesion development in lit/lit mice was age, as lit/lit males killed at 32 weeks of age had smaller volume fractions of foci than any group of lit/lit mice killed at 50 weeks of age. Thus, preneoplastic foci grow in lit/lit mice, but their development is refractory to modulation by genetic background, castration and possibly ovariectomy.

All the lit/lit groups at 50 weeks of age had appreciable numbers of small basophilic preneoplastic lesions and they rarely developed adenomas. We observed that at 12 days of age (the age at which mice are treated with DEN), there are no differences between B6 wild-type and B6-lit/lit mice in the metabolic activation of DEN and there is little difference in proliferation in the liver. Furthermore, lit/lit mice developed significant numbers of preneoplastic foci, but they apparently grew more slowly than in normal mice. Thus, in this model, growth hormone is not likely to affect initiation per se. Wild-type levels of growth hormone presumably affect the promotion stage of hepatocarcinogenesis by increasing the net growth rate of preneoplastic lesions.

The most outwardly apparent effects of the little mutation are the smaller stature and lower body mass of homozygous animals; lit/lit mice in our experiments were ~45% smaller by weight than wild-type mice (data not shown). Dietary restriction, which has been shown in several protocols to inhibit liver tumorigenesis (6870), is not the cause of suppressed tumor development in lit/lit mice, because B6-lit/lit mice eat as much as wild-type mice in proportion to body weight (43). Although mice with lower body weights generally have lower incidences of liver neoplasms, body weight reduction does not decrease the sex difference in tumor incidence. In fact, larger sex differences in tumor incidence have been reported in low body weight animals and the sex difference is diminished in the high body weight animals (71). Thus, the abrogation of the sex difference in lit/lit mice suggests that the effect of growth hormone deficiency is not simply a result of body weight reduction. On the other hand, the mechanisms by which caloric restriction retards cancer development may be related to alterations in hormone and growth factor levels (72) and it is possible that part of the effect of caloric restriction on reducing the incidence of liver neoplasms is mediated by growth hormone. There is evidence from studies on several species that caloric restriction alters growth hormone levels, although the effects vary with age and the degree of restriction (73,74).

The little mutation affects pituitary function and specifically it causes resistance to growth hormone releasing hormone. Other than growth hormone deficiency, normal serum levels of other pituitary hormones have been reported in B6-lit/lit mice (38,39). Consequently, the effect of the little mutation on susceptibility to hepatocarcinogenesis is specific to the deficiency in growth hormone. We do not know, however, whether the effect is direct: that is, whether growth hormone binding to the growth hormone receptor on a hepatocyte increases the chances of that particular hepatocyte forming a tumor. One could test this hypothesis directly using growth hormone receptor knockout mice (75) and a chimeric mouse approach similar to the one showing the cell autonomous effects of the C3H and BR susceptibility loci (65,66).

The effect of growth hormone on tumorigenesis could be mediated by intracellular as well as extracellular factors. Growth hormone activates Stat-1, -3, -5a and -5b in hepatocytes (76,77), but Stat-5a and -5b are responsible for most of the growth-promoting effects. Stat-5b is activated by pulsatile growth hormone release and is responsible for growth hormone-dependent masculinization in the liver (78,79). Similarly, activation of Stat-1 and -3 is non-linear with respect to growth hormone concentrations. Both of these Stat factors interact with the Fos promoter (76) and growth hormone regulation of c-fos and c-myc may play a role in sex-differentiated hepatocarcinogenesis (80). A parallel intracellular pathway stimulated by growth hormone involves Jak-2 phosphorylation of the epidermal growth factor receptor (Egfr) and subsequent activation of the MAP kinase cascade (81). Egfr levels are also up-regulated by growth hormone in the liver (82,83) and transgenic mice overexpressing the Egfr ligand TGF-{alpha} develop spontaneous hepatocellular carcinoma (84). Another growth factor, Insulin-like growth factor-1 (Igf-1) and its regulator, the Igf binding protein acid-labile subunit, are produced in the liver in response to growth hormone and Igf-1 mediates certain growth promoting effects of growth hormone (43,85). Thus, the complete effect of growth hormone on tumor development may require multiple intracellular and subsequent extracellular signals.

In summary, using perinatal treatment with DEN as a mouse model of hepatocarcinogenesis, our studies show that growth hormone is among the most potent known endogenous regulators of susceptibility. Moreover, the absence of growth hormone abrogates modification of susceptibility by sex hormones and genetic background. Our results do not prove, but are consistent with, the hypothesis that tumor promotion by testosterone is mediated by growth hormone. In addition, strain differences in susceptibility require wild-type levels of growth hormone. However, there is strong evidence against strain differences in serum levels of growth hormone being responsible for differences in susceptibility among C3H, BR and B6 inbred mice. The possibility exists that the molecular pathways activated by growth hormone intersect with the strain-dependent factors that control susceptibility in the target hepatocyte. On the other hand, growth hormone deficiency may simply limit the rate of preneoplastic focus growth to a level where strain-dependent factors, which are known to affect focal growth rates, have no effect. In such a case, the molecular pathways may be entirely independent. Which of these scenarios is correct will likely remain unclear until the molecular identities of the susceptibility loci in BR and C3H mice are resolved.


    Notes
 
1 Present addresses: Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112 and Back

2 Department of Biology, Georgia State University, Atlanta, GA 30303, USA Back

3 To whom correspondence should be addressed Email: drinkwater{at}oncology.wisc.edu Back


    Acknowledgments
 
We thank Jane Weeks and Harlene Edwards for their technical assistance; Henry Pitot for providing histopathological diagnoses and Eric Sandgren, Brad Clark, Herb Chen, Jennifer Drew, Andrea Bilger and Sue Schadewald for critical reviews of the manuscript. This work was supported by grants CA07175, CA09135 and CA22484 from the National Cancer Institute and J.M.B was supported by funds from the Cremer Foundation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 17, 2001; revised July 10, 2001; accepted July 20, 2001.