The role of reactive oxygen species in cisplatin-induced apoptosis in human malignant testicular germ cell lines.

Testicular germ cell tumours (TGCT) are the most common solid tumour among young males. Whereas in 1970s, only 5% of patients with a metastatic testicular tumours survived their disease, these days 80% of patients treated by modern cisdiamminedichloroplatinum (cisplatin, CDDP)-based chemotherapy are cured. Although data are accumulating on the effect of the mitogen-activated protein kinase (MAPK) family on the CDDP-induced apoptosis in tumour cells, the mechanisms by which CDDP initiates apoptosis in TGCT are not completely understood. Applying Western blot and phosphorylated kinase-specific ELISA analyses, flow cytometry, blocking experiments, and morphological methods we sought here to define the MAPK pathway(s) involved in the CDDP-induced apoptosis in the human TGCT cell line NCCIT. Our experiments showed that within hours of CDDP application only the extracellular signal-regulated kinase (ERK) was dually phosphorylated and caspase-3 became active. Functional assays using chemical inhibitors demonstrated that the phosphorylation of ERK was mediated by reactive oxygen species in an Raf-1-independent manner and required the activation of caspase-3. Thus, our data suggest that CDDP mediates its apoptosis-inducing effect on the human malignant testicular germ cells, at least partially, through activation of the MEK-ERK signaling pathway in a ROS-dependent, Raf-1-independent manner.

Cisplatin-induced apoptosis in human malignant testicular germ cell lines depends on MEK/ERK activation.

Testicular germ cell tumours (TGCT) represent the most common malignancies in young males. Whereas in 1970s, the survival rate in patients with metastatic testicular tumours was only 5%, these days, 80% of the patients treated by modern chemotherapy will survive their disease. The drug that revolutionised the cure rate for patients with metastatic testicular tumours was cisdiamminedichloroplatinum (cisplatin, CDDP). In vitro experiments on neoplastic germ cell lines showed that their exquisite sensitivity to CDDP could be attributed to p53-dependent and -independent pathways. Applying cDNA macroarray, semiquantitative RT-PCR and Western blot analyses, blocking experiments, caspase activity assays, and morphological methods, we sought here to define the p53-independent pathway(s) involved in the CDDP-induced apoptosis. For this purpose, we used the human TGCT cell line NCCIT, the mutated p53 of which is known to remain inactive during the course of CDDP-induced apoptosis. Our experiments showed that within hours of CDDP application, two prototype members of the 'mitogen-activated protein kinase' (MAPK) family, designated 'MAPK ERK kinase' (MEK) and 'extracellular signal-regulated kinase' (ERK), were dually phosphorylated and caspase-3 became active. Functional assays using MEK inhibitors demonstrated that the phosphorylation of MEK and ERK was required for the activation of caspase-3 as the executing caspase. Interestingly, experiments with the human malignant germ cell line NTERA, which is known to possess wild-type p53, revealed the same results. Thus, our data suggest that CDDP mediates its p53-independent apoptosis-inducing effect on the malignant human testicular germ cells--at least partially--through activation of the MEK-ERK signalling pathway. July 2004

High-performance liquid chromatographic determination of 3-hydroxy-3-methylglutaryl coenzyme A as diphenacyl ester of 3-hydroxy-3-methylglutarate.

A method for derivatization of (S)-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) by phenacylation in tetrahexylammonium hydrogensulphate as phase-transfer reagent and high-performance liquid chromatography of the HMG-diphenacyl ester is described. To avoid interference with buffers and other ions used to dissolve biological material, the alkaline hydrolysate of CoA esters has to be neutralized with phosphoric rather than hydrochloric acid. The latter reacts with phenacyl bromide. The pH during phenacylation has to be maintained in the range 6-7 to provide sufficient dissociation of HMG.

Plastidic Isoprenoid Synthesis during Chloroplast Development : Change from Metabolic Autonomy to a Division-of-Labor Stage.

The chloroplast isoprenoid synthesis of very young leaves is supplied by the plastidic CO(2) --> pyruvate --> acetyl-coenzyme A (C(3) --> C(2)) metabolism (D Schulze-Siebert, G Schultz [1987] Plant Physiol 84: 1233-1237) and occurs via the plastidic mevalonate pathway. The plastidic C(3) --> C(2) metabolism and/or plastidic mevalonate pathway of barley (Hordeum vulgare L.) seedlings changes from maximal activity at the leaf base (containing developing chloroplasts with incomplete thylakoid stacking but a considerable rate of photosynthetic CO(2)-fixation) almost to ineffectivity at the leaf tip (containing mature chloroplasts with maximal photosynthetic activity). The ability to import isopentenyl diphosphate from the extraplastidic space gradually increases to substitute for the loss of endogenous intermediate supply for chloroplast isoprenoid synthesis (change from autonomic to division-of-labor stage). Fatty acid synthesis from NaH(14)CO(3) decreases in the same manner as shown for leaf sections and chloroplasts isolated from these. Evidence has been obtained for a drastic decrease of pyruvate decarboxylase-dehydrogenase activity during chloroplast development compared with other anabolic chloroplast pathways (synthesis of aromatic amino acid and branched chain amino acids). The noncompetition of pyruvate and acetate in isotopic dilution studies indicates that both a pyruvate-derived and an acetate-derived compound are simultaneously needed to form introductory intermediates of the mevalonate pathway, presumably acetoacetyl-coenzyme A.