The mRNA species for prolactin receptor (PRL-R) isoforms, long and short form PRL-Rs, were estimated by the reverse transcription-polymerase chain reaction method in the rat brain (cerebrum) during the oestrous cycle, pregnancy and lactation. The levels of long form PRL-R mRNA increased at pro-oestrus and oestrus, at the same time as serum prolactin levels increased, whereas the mRNA level of short form PRL-R was relatively unchanged. Long form PRL-R mRNA expression was also markedly increased in the brain at mid- and late gestation, and this elevated mRNA level was maintained during the period of lactation. In contrast, basal levels of short form PRL-R mRNA were also maintained throughout these periods of gestation and lactation. Ovariectomy moderately reduced brain mRNA levels of both long and short form PRL-R from the levels of those in control dioestrous rats, and hypophysectomy further suppressed them to the lowest levels. Administration of oestradiol valerate (E2V) or 17 alpha-hydroxyprogesterone caproate (17OHPC) to ovariectomized rats resulted in dramatic increases in long form PRL-R mRNA levels in the brain, whereas no significant increase in short form PRL-R mRNA was observed. In rats which were ovariectomized and hypophysectomized, the administration of 17OHPC, rat prolactin or rat GH partially restored the brain level of long form PRL-R mRNA but not short form PRL-R mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactive oxygen species (ROS) are constantly generated in cells by several biological metabolic processes. A moderate level of intracellular ROS is thought to be important as a stimulant of cellular proliferation. However, the overproduction of ROS can result in detrimental cellular damage, including lipid peroxidation, DNA damage, protein oxidation and enzyme inactivation, which can ultimately lead to cell death.1,2) Aerobic cells contain several antioxidant enzymes, such as superoxide dismutase (SOD), catalase and various peroxidases, to maintain appropriate intracellular ROS levels and to prevent against oxidative damage. Generally, the cellular SOD activity appeared to be a little lower in malignant tissues than in normal tissues.3) 2-Methoxyestradiol (2-ME), an endogenous metabolite of 17b-estradiol, is present in human blood and urine. 2-ME is produced by 2-hydroxylation and subsequent O-methylation 4) and is known to have a low binding activity to the estrogen receptor.5) 2-ME has been shown to have potent anti-proliferative effects on various types of human cancer cells, including leukaemia, multiple myeloma, prostate cancer, gastric carcinoma and hepatoma.6-10) 2-ME has been reported to induce an accumulation of O 2 · Ϫ by inhibiting SOD, and to make G 2 /M-phase arrest accompanied by the inhibition of tubulin polymerization. [6][7][8][9][10] This compound is currently in clinical trial in the United States.8) Buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, has been used in clinical trials of cancer treatment. 11,12) However, the combination of the effect of 2-ME and the modification of ROS generation and the metabolic system have not been investigated in renal carcinoma cell lines.Renal carcinoma is one of the drug resistant malignancies in humans. ACHN is a cell line of human renal cell carcinoma, and ACVB is a vinblastine-resistant cell line of ACHN. ACHN has been used to study cytotoxic effects of several anticancer agents. For example, synergistic cytotoxicity by the combination of anti-Fas monoclonal antibody with adriamycin in ACHN had been reported. 13) In the present study, we examined the effect of combination with 2-ME, BSO and hydrogen peroxide (H 2 O 2 ) on the viability of human renal carcinoma cell lines (ACHN and ACVB). We have also analyzed the relationship between cell viability and intracellular ROS levels. MATERIALS AND METHODSChemicals 2-ME, estradiol, dihydroethidium (HEt), 2Ј,7Ј-dichlorofluoresin diacetate (DCFH-DA) and buthionine sulfoximine were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). RPMI-1640 was obtained from Invitrogen (Rockvill, MD, U.S.A.). Vinblastine, H 2 O 2 and 5Ј,5Ј-dithiobis 2-nitrobenzoic acid (DTNB) were obtained from Wako Pure Chemicals (Osaka). A WST-1 cell counting kit was obtained from Dojindo (Kumamoto). Fetal bovine serum (FBS) was obtained from ICN (Aurora, OH, U.S.A.).Cell Line and Cell Culture Human renal carcinoma cell line (ACHN) and its vinblastine resistant cell line (ACVB) were received from Prof. Hirai of Kobe ...

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