In previous studies hepatocytes undergoing cell death by apoptosis but not normal hepatocytes in rat liver showed immunostaining for transforming growth factor ,1 (TGF-13). Staining was much stronger with antibodies recognizing the pro-region of TGF-I31 than the mature peptide itself. Therefore we investigated the ability of both forms of TGF-31 to induce apoptosis in primary cultures of rat hepatocytes.Mature TGF-fi1 induced rounding up of the cells and fragmentation into multiple vesicles. As revealed by the DNAspecific stain H33258, the chromatin of these cells condensed and segregated into masses at the nuclear membrane; this was obviously followed by fragmentation of the nucleus. Ultrastructurally the cytoplasm was well preserved, as demonstrated by the presence of intact cell organelles. These features strongly suggest the occurrence of apoptosis. Quantification of nuclei with condensed chromatin revealed that mature TGF-131 was 30-fold more effective than the TGF-P1 latency-associated protein complex. Finally, we administered TGF-131 in vivo using an experimental model in which regression of rat liver was initiated by a short preceding treatment with the hepatomitogen cyproterone acetate. Two doses of TGF-P1, each 1 nM/kg, augmented the incidence of apoptotic hepatocytes 5-fold. Equimolar doses of TGF-fi1 latency-associated protein complex were ineffective. These studies suggest that TGF-P1 is involved in the initiation of apoptosis in the liver and that the mature form of TGF-fi1 is the active principle.
Abstract. Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis.Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti-PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34 ~2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase.The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34 ~c2 kinase.OPTOSIS waS originally defined as a program of morphological changes accompanying cell death during which the cells round up and the chromatin condenses, leading to the formation of crescent-shaped masses aggregating at the membrane. In parallel the nucleolus dissolves (Kerr, 1971). These initial changes are followed by cytoplasmic and nuclear compaction, and ultimately by fragmentation of the cell. The nuclear fragments formed in this process are still surrounded by the nuclear membranes (Kerr, 1971;Oberhammer et al., 1993a). As a common biochemical marker for apoptosis, an activation of a nonlysosomal, Ca2+/MgZ÷-dependent endonuclease has been suggested (Wyllie, 1980;Cohen and Duke, 1984). The response is so readily identifiable that endonuclease activation
In previous studies we showed that transforming growth factor-beta 1 induces apoptosis in hepatocyte cultures and regressing livers, the mature form being more potent than the transforming growth factor-beta 1 latency-associated protein. In this study we addressed the question of whether apoptosis can be induced within a short time after administration of transforming growth factor-beta 1. Five hours after a single intravenous injection of 25 micrograms mature transforming growth factor-beta 1/kg body weight, apoptosis is augmented ninefold in the regressing rat liver. A second preceding application induces no further augmentation. Transforming growth factor-beta 1 latency-associated protein shows no effect with either regimen. Morphological evaluation shows that 5 hr after injection of transforming growth factor-beta 1 nearly all apoptotic bodies are already engulfed by their neighbor cells. After homogenization of the transforming growth factor-beta 1-treated livers, the condensed apoptotic bodies are not destroyed and remain in the nuclear pellet. No DNA fragmentation into oligosomes could be detected after purification of the DNA from the nuclear pellet and application to conventional gel electrophoresis. Application of in situ nick translation, which allows detection of DNA single- and double-strand breaks in individual apoptotic bodies, also revealed no substantial fragmentation of the DNA in apoptotic bodies. These studies show that transforming growth factor-beta 1 is able to induce apoptosis within a rather short time and also suggest that in vivo digestion of the DNA does not lead to chromatin condensation.
Recently, cases of liver damage and liver tumors have been reported after treatment of prostate cancer patients with the antiandrogen cyproterone acetate (CPA). In rat liver, CPA initiates a wave of DNA synthesis that is accompanied by apoptosis. In apoptotic hepatocytes, a latent form of transforming growth factor beta 1 (TGF-beta 1) is detectable by immunohistochemistry. Injection of a single dose of TGF-beta 1 induces apoptosis in the liver of animals pretreated with CPA but has an insignificant effect in untreated animals. In this study, we show by Northern analysis that there is increased expression of TGF-beta 1 in the liver after CPA treatment. Detection of TGF-beta 1 with in situ hybridization showed that TGF-beta 1 was synthesized in the parenchymal cells. Time course and dose-response experiments performed 48 hours after the last application of CPA showed that apoptotic nuclei with chromatin condensed at the nuclear periphery (AN) were already visible 2 hours after injection (0.13%), and apoptotic bodies (ABs) increased 2 to 9 hours after the injection (from 1.28% to 6.67%) after 25 micrograms TGF-beta 1/kg. At 4.5 hours after injection, an induction of apoptosis could be detected with 0.25 microgram TGF-beta 1/kg and after the maximum dose (250 micrograms TGF-beta 1/kg) ANs (0.24%) and ABs (16.74%) were homogeneously distributed throughout the liver lobe. Irrespective of the dose or time after injection of TGF-beta 1, 82% of the ABs were localized within hepatocytes. Liver enzymes were detected in high amounts in the serum (eightfold elevation of glutamate dehydrogenase, fivefold elevation of alanine transaminase [ALT]) 7 hours after the first visible sign of apoptosis. After an additional 20 hours, the liver contained many necrotic figures. These results suggest that the combination of TGF-beta 1 expression coupled with a strikingly enhanced sensitivity to the induction of apoptosis could be responsible both for the liver damage and the development of liver tumors observed after treatment with CPA.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.