Paradoxical effects of platelet-derived growth factor-A overexpression in malignant mesothelioma. Antiproliferative effects in vitro and tumorigenic stimulation in vivo.

Malignant mesothelioma is associated with asbestos exposure and remains resistant to all therapeutic intervention. Previous studies have suggested an enhancing role for platelet-derived growth factor (PDGF) in mesothelial tumorigenicity, although the mechanism by which PDGF facilitates tumorigenicity is unknown. Here, we evaluate the contribution of PDGF-A expression to mesothelial tumorigenicity using ectopic modulation of PDGF-A expression. We find, in accordance with other reports, that the receptor for PDGF-A, although expressed at high levels in normal human mesothelial cells, is not easily detectable in mesothelioma. Further, we show that PDGF-A overexpression is responsible for autocrine downregulation of its receptor. Our data indicate, surprisingly, that for mesothelioma cells in vitro, high-level activation of a PDGF-A-PDGF receptor loop is antiproliferative whereas abrogation of PDGF-A expression stimulates growth. These data suggest that PDGF-A does not contribute to tumorigenicity by autocrine stimulation of proliferation. In contrast, increased PDGF-A expression in vivo increases tumor incidence and growth rate and decreases the latency period to tumor formation whereas abrogation of PDGF-A expression decreases tumor incidence and increases latency. Thus, the tumorigenic effect of PDGF-A must act through paracrine mechanisms relevant at early stages of tumor initiation.

Loss of nuclear p53 protein in preneoplastic rat hepatocytes is accompanied by Mdm2 and Bcl-2 overexpression and by defective response to DNA damage in vivo.

Previous studies have indicated that isolated preneoplastic rat hepatocytes in vitro fail to induce nuclear p53 protein and fail to block replication in response to genotoxic compounds. This suggests that defects in the protection of genomic integrity are part of their premalignant character. In the present study, we have investigated if similar defects occur in vivo. Preneoplastic glutathione-S-transferase (GST) 7-7-positive foci were induced in male Wistar rats by diethylnitrosamine (DEN) initiation and promotion with 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PH). The response to genotoxic damage was studied by X-irradiation. p53 protein was moderately expressed in nuclei in surrounding hepatocytes. This nuclear p53 staining had decreased 2 weeks after 2-AAF treatment. In foci, the protein was detected in the cytoplasm whereas the nuclei were negative. Levels of p21(waf1/cip1) protein were high in nuclei and cytoplasm of surrounding hepatocytes, whereas the expression in foci was low. A low level of Mdm2 in nuclei was observed in surrounding liver, while both Mdm2 and Bcl-2 protein were strongly expressed in the cytoplasm in foci. X-ray exposure further induced nuclear expression of p53, p21(waf1/cip1), and Mdm2 in surrounding hepatocytes, but focal nuclei were still negative. DNA replication was strongly reduced by X-irradiation in surrounding hepatocytes, but only partially reduced in the foci. These results indicate that the p53 pathway of response to genomic stress is impaired in preneoplastic cells in vivo. This may support their clonal expansion and their further malignant transformation because protection against genetic damage is diminished.

Lack of p53 protein expression in preneoplastic rat hepatocytes in vitro after exposure to N-acetoxy-acetylaminofluorene, X-rays or a proteasome inhibitor.

Clonal expansion of initiated cells is an important process in carcinogenesis. Loss of functional p53 protein in initiated, preneoplastic cells might be involved in this process because such a loss would favour cell growth at the expense of normal cells upon exposure to genotoxic compounds. We have tested the hypothesis that p53 is not expressed in preneoplastic cells in the rat liver. Hepatocytes were isolated from livers of 10-week-old female rats that contained foci of preneoplastic hepatocytes, generated by 6-7 weekly injections of diethylnitrosamine (0.15 mmol/kg body wt intraperitoneally (i.p.)), starting 24 h after birth. The mixture of phenotypically normal and preneoplastic hepatocytes was exposed to X-rays or N-acetoxy-acetylaminofluorene (NAAAF), both causing DNA damage directly. At 24 and 48 h after exposure the cells were fixed and double stained for glutathione-S-transferase 7-7 (GST7-7), to identify preneoplastic cells, and p53. The percentage of p53-positive cells was much lower in GST7-7 positive (GST7-7+) than in GST7-7 negative (GST7-7-) hepatocytes. Exposure of cells to X-rays or NAAAF induced p53 in GST7-7- cells after 24 h, but GST7-7+ hepatocytes failed to do so. These results suggest that preneoplastic cells do not express p53 or have an attenuated p53 response to genotoxic treatments. This was confirmed when the cells were exposed to a proteasome inhibitor, PSI, which inhibits p53 degradation: a 12-fold increase in p53-positive cells was found after 48 h in GST7-7- hepatocytes, but in GST7-7+ hepatocytes no increase was observed. The percentage of GST7-7+ hepatocytes among surviving cells was increased after exposure to NAAAF, suggesting that these are more resistant to NAAAF than GST7-7- cells. This was not observed with PSI. These results indicate that preneoplastic hepatocytes have a lower p53 protein content and are not able to increase p53 upon inhibition of p53 breakdown or upon induction of DNA damage. Therefore, loss of p53 may favour clonal expansion of preneoplastic hepatocytes in the rat after administration of hepatocarcinogens or X-rays.

Phosphorescent platinum/palladium coproporphyrins for time-resolved luminescence microscopy.

Streptavidin and antibodies were labeled with phosphorescent platinum and palladium coproporphyrin. The optimal conjugates were selected on the basis of spectroscopic analysis (molar extinction coefficient, quantum yield, lifetime) and using ELISA assays to determine the retention of biological activity and immunospecificity. They were subsequently tested for the detection of prostate-specific antigen, glucagon, human androgen receptor, p53, and glutathione transferase in strongly autofluorescent tissues. Furthermore, platinum and palladium coproporphyrin-labeled dUTPs were synthesized for the enzymatic labeling of DNA probes. Porphyrin-labeled DNA probes and porphyrin-labeled streptavidin conjugates were evaluated for DNA in situ hybridization on metaphase spreads, using direct and indirect methods, respectively. The developed in situ detection technology is shown to be applicable not only in mammals but also in plants. A modular- based time-resolved microscope was constructed and used for the evaluation of porphyrin-stained samples. The time-resolved module was found suitable for detection of antigens and DNA targets in an autofluorescent environment. Higher image contrasts were generally obtained in comparison with conventional detection systems (e.g., fourfold improvement in detection of glutathione transferase).

Immunohistochemical visualization of wild-type p53 protein in paraffin-embedded rat liver using tyramide amplification: zonal hepatic distribution of p53 protein after N-hydroxy-2-acetylaminofluorene administration.

P53 protein plays an important role in regulation of the cell cycle. Recently, a role in tumour genesis has also been suggested. The protein is induced after various forms of DNA damage. Immunohistochemical detection of p53 protein showed positive cells in human skin after UV-irradiation, in mouse skin after benzo[a]pyrene treatment and in mouse spleen, thymus and bone after gamma-irradiation. However, no staining was found in mouse and rat liver with traditional immunohistochemical staining methods due to the low amount of p53 present. This seriously hampered studies on the role of p53 in hepatocarcinogenesis. We have developed a more sensitive immunohistochemical method for staining of p53 in paraffin-embedded sections of rat liver using microwave irradiation for antigen retrieval, avidin-biotin complexing and tyramide amplification. A strong, specific fluorescence signal for p53 was found in hepatocytes of rats that had received the hepatocarcinogen N-hydroxy-2-acetylaminofluorene; in control liver no such p53 staining was observed. The fluorescence was located in the nucleus of hepatocytes in zone 1 of the liver. This agrees with the fact that N-hydroxy-2-acetylaminofluorene causes cytotoxicity in this zone.

p53 protein expression by hepatocarcinogens in the rat liver and its potential role in mitoinhibition of normal hepatocytes as a mechanism of hepatic tumour promotion.

The tumour suppressor gene p53 is expressed in response to DNA-damage; its protein product blocks cells in the G1-phase of the cell cycle. This gives cells additional time to repair their DNA-damage. However, it may trigger apoptosis if damage is too high. Loss of p53 function appears to be an important step in carcinogenesis because 50% of human tumours have lost functional p53. In order to study the role of p53 in experimental hepatocarcinogenesis, we determined the expression of p53 in rat liver in response to various hepatocarcinogenic and hepatotoxic compounds. Administration of hepatocarcinogenic compounds increased p53 protein levels in the liver as detected by immunoprecipitation followed by SDS-PAGE and Western blotting with ECL-detection. The hepatocarcinogens included N-hydroxy-2-acetylaminofluorene, aflatoxin B1, and diethylnitrosamine. Their structural analogues N-hydroxy-4-acetylaminobiphenyl and ethyl methane-sulphonate which are not hepatocarcinogenic, did not induce p53. Also, two hepatotoxic compounds (carbon tetrachloride, D-galactosamine) did not induce p53. Other compounds that induced p53 in the rat liver were 2-aminofluorene (administered by drinking water for two weeks) and tris-(2,3-dibromopropyl)phosphate. Benzo[a]pyrene did not induce p53. N-Hydroxy-2-acetylaminofluorene, aflatoxin B1, and diethylnitrosamine are potent hepatic tumour promoters. At the same time, they induce p53 protein expression and inhibit proliferation of normal hepatocytes. Because this is not observed with non-hepatocarcinogenic analogues, it suggests an involvement of p53 expression in hepatic tumour promotion. A possible mechanism is discussed.