Inherent growth advantage of (pre)malignant hepatocytes associated with nuclear translocation of pro-transforming growth factor alpha

Abstract

The pro-peptide of transforming growth factor alpha (proTGFalpha) was recently found in hepatocyte nuclei preparing for DNA replication, which suggests a role of nuclear proTGFalpha for mitogenic signalling. This study investigates whether the nuclear occurrence of the pro-peptide is involved in the altered growth regulation of (pre)malignant hepatocytes. In human hepatocarcinogenesis, the incidence of proTGFalpha-positive and replicating nuclei gradually increased from normal liver, to dysplastic nodules, to hepatocellular carcinoma. ProTGFalpha-positive nuclei almost always were in DNA synthesis. Also, in rat hepatocarcinogenesis, proTGFalpha-positive nuclei occurred in (pre)malignant hepatocytes at significantly higher incidences than in unaltered hepatocytes. For functional studies unaltered (GSTp(-)) and premalignant (GSTp(+)) rat hepatocytes were isolated by collagenase perfusion and cultivated. Again, DNA synthesis occurred almost exclusively in proTGFalpha-positive nuclei. GSTp(+) hepatocytes showed an approximately 3-fold higher frequency of proTGFalpha-positive nuclei and DNA replication than GSTp(-) cells. Treatment of cultures with the mitogen cyproterone acetate (CPA) elevated the incidence of proTGFalpha-positive nuclei and DNA synthesis in parallel. Conversely, transforming growth factor beta1 (TGFbeta1) lowered both. These effects of CPA and TGFbeta1 were significantly more pronounced in GSTp(+) than in GSTp(-) hepatocytes. In conclusion, nuclear translocation of proTGFalpha increases in the course of hepatocarcinogenesis and appears to be involved in the inherent growth advantage of (pre)malignant hepatocytes.

Figure 1

Occurrence of nuclear proTGFα in human and rat hepatocarcinogenesis. (A–C) Rat liver 12 months post-NNM: serial sections of a preneoplastic lesion stained for GSTp (A) and TGFα (B); (C) HCC with proTGFα-pos nuclei (C). (D) Preneoplastic GSTp⁺ hepatocytes (violet) with proTGFα-pos nucleus (brown) and incorporated ³H-thymidine (black spots) in primary culture; hepatocytes were isolated at day 21 post-NNM and cultured for 48 h; ³H-thymidine was added to the medium 24 h before harvesting. (E) Human HCC and (F) human HepG2-hepatoma cells with proTGFα-pos nuclei. Magnifications: × 50 for (A), (B), × 75 for (E); × 25 for (C); × 200 for (D, F).

Figure 2

Detection of proTGFα in the nuclear fraction of human HepG2-hepatoma cells and of rat liver. Nuclear matrix proteins of hepatoma cells (A) and of an untreated male rat liver (B) were subjected to two-dimensional immunoblotting. The gels covered a range of pI 3–8 and Mr 4–40 kDa. (A) Spots 1–4 were analysed by maldi-tof: spot 1 was identified as wild-type proTGFα; ^* indicates the amino-acid sequence identified, which covers 19% of the total pro-peptide of TGFα.

Figure 3

Increased cell replication and incidences of proTGFα-pos nuclei in human and rat hepatocarcinogenesis. White columns represent the percentage of nuclei in S-phase (LI%), dark columns the percentage of proTGFα-pos nuclei. Humans: three hepatocellular adenomas, nine dysplastic nodules, 10 heptocellular carcinomas and the surrounding liver of the 22 cases were evaluated. At least 1000 hepatocyte nuclei (on average 2055 nuclei) per liver or tumour were screened for Ki-67 and proTGFα positivity. Rats: time interval between ³H-thymidine injection and killing was 36 h. The percentages of proTGFα-pos nuclei and LI were determined at least in: 2000 nuclei of GSTp⁻ and 1221 nuclei of GSTp⁺ hepatocytes per liver (n=13); 1381 nuclei per adenoma (n=15); 1362 nuclei per carcinoma (n=14). nl, normal liver; pr, preneoplastic GSTp⁺ lesion; ad, hepatocellular adenoma; dn, dysplastic nodule; ca, HCC. Incidences are given; vertical lines give 95% confidence intervals: no overlap indicates a statistically significant difference for P<0.05.

Figure 4

DNA synthesis (LI(%)) and % of proTGFα-pos nuclei in cultured GSTp⁻ and GSTp⁺ hepatocytes. ³H-thymidine was added 24 h before harvesting of cells. The % of proTGFα-pos nuclei was determined after 48 h of culture. Symbols: Δ or open columns, GSTp⁻ cells; ▪ or dark columns, GSTp⁺ cells. In each of the experiments, 2000 nuclei of GSTp⁻ cells and 600 nuclei of GSTp⁺ cells were evaluated. Means±s.d. are given from separate experiments with cultures from five rats. Statistics of LI(%) in GSTp⁻ vs GSTp⁺ cells over time-course by Kruskal–Wallis test: (a) P<0.001. Statistics of LI(%) in GSTp⁻ cells vs GSTp⁺ cells at the last time point of evaluation by Wilcoxon's test: (b) P<0.01. Statistics of % of proTGFα-pos nuclei in GSTp⁻ vs GSTp⁺ cells by Wilcoxon's test: (c) P<0.001.

Figure 5

Mature TGFα increases the percentage of nuclei in S-phase (LI%) preferentially in GSTp⁻ hepatocytes without nuclear proTGFα. ³H-thymidine was added 24 h before harvesting of cells. The percentages of replicating and of proTGFα-pos nuclei were determined after 48 h of culture. For the number of experiments and cells scored, see Figure 4. Hatched portions of the bars indicate LI of proTGFα-pos nuclei; nonhatched portions of the bars indicate LI of proTGFα-neg nuclei; the sum of the hatched and unhatched portion (total bar) indicates LI of all nuclei; Co: DMSO-control; TGFα: mature TGFα; Tyr: Tyrphostin A25. Statistics by Student's t-test: (a) P<0.05.

Figure 6

Transforming growth factor β1 reduces DNA synthesis and proTGFα-pos nuclei in GSTp⁻ and GSTp⁺ cells cultured for 48 h. ³H-thymidine was added 24 h before harvesting of cells. Symbols: Δ, GSTp⁻ cells, ▪, GSTp⁺ cells. Data are calculated as fold control; absolute values are given in Table 1; number of experiments and cells scored see Figure 4. Means±s.d. are given. Statistics by Kruskal–Wallis test for dose–response effects within a cell population: (a) P<0.01; (b) P<0.05.

Figure 7

Cyproterone acetate induces DNA synthesis and proTGFα-pos nuclei in GSTp⁻ and GSTp⁺ cells cultured for 48 h. ³H-thymidine was added 24 h before harvesting of cells. Symbols: light columns, GSTp⁻ cells; dark columns, GSTp⁺ cells. For the number of experiments and cells scored, see Figure 4. Means±s.d. are given. Statistics by Wilcoxon's test for Co vs CPA: (a) P<0.01; (b) P<0.05.