Yoshiaki Onishi

Topoisomerase inhibitors induce apoptosis in thymocytes

The effects of the inhibitors of topoisomerase I and II, camptothecin and etoposide, as well as novobiocin and adriamycin, on the DNA fragmentation and viability of mouse thymocytes in primary culture were examined. All inhibitors were shown to produce dose-dependent internucleosomal DNA cleavage by resolving isolated DNA by agarose-gel electrophoresis. The DNA fragmentation seemed to precede cell death, determined on the basis of LDH release, by a few hours. Etoposide-induced DNA fragmentation progressively increased after incubation and was enhanced by pretreatment with phorbol 12,13-dibutyrate, a phorbol ester capable of activating protein kinase C, whereas camptothecin-induced DNA fragmentation increased progressively after 12 h incubation and was unaffected by phorbol 12,13-dibutyrate-pretreatment. The process was also energy-dependent and required RNA and protein synthesis and protein phosphorylation, since it was inhibited by sodium azide, actinomycin D, cycloheximide and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine hydrochloride, a protein kinase inhibitor. DNA fragmentation was also inhibited by zinc ions, suggesting the involvement of a specific endonuclease in DNA cleavage. These phenomena are similar to those detected in thymocytes undergoing apoptosis following exposure to glucocorticoids (Cohen, J.J. and Duke, R.C. (1984) J. Immunol. 132, 38-42). Considering that topoisomerases function in cellular proliferation and differentiation by altering DNA topology, the results suggest that topoisomerases have important roles in T-lymphocyte ontogeny in the thymus and are in part involved in the elimination of autoreactive or harmful cells by an apoptotic process.

Topoisomerase II inhibitor-induced apoptosis in thymocytes and lymphoma cells

DNA topoisomerase II is a nuclear enzyme that modulates DNA topology during several metabolic processes and is the target of several antitumor drugs. The primary effect of anticancer agents is to induce apoptosis. The present study showed that etoposide, a topoisomerase II inhibitor which forms cleavable complexes, induced apoptosis in nonproliferative thymocytes and proliferative RVC cells, whereas ICRF-154, a bis(2,6-dioxopiperazine) derivative which does not form a cleavable complex, induced apoptosis only in thymocytes. Both etoposide and ICRF-154 inhibited topoisomerase II activity in thymocytes and RVC cells to a similar extent. Etoposide had no effect on the cell cycle of RVC cells, but ICRF-154 induced cell cycle arrest at the G2/M stage followed by cell death without forming a DNA ladder on an agarose gel. Incubation with ICRF-154 reduced the expression of topoisomerase IIa in thymocytes and IIb in RVC cells. These findings suggest that the catalytic inhibitor, ICRF-154, has a mechanism of cytotoxicity which differs from that of etoposide. In RVC cells exposed to etoposide, we identified two clones that were suppressed early in the incubation. One was highly homologous to hnRNP A1 which modulates splicing of selected transcripts or stabilizes mRNAs. The other was a novel gene of which the function remains unknown. These genes were also altered in RVC cells exposed to camptothecin, which underwent apoptosis, but not in those incubated with ICRF-154, indicating that the suppression of these genes is related to inhibitor-induced DNA breaks resulting in apoptosis. In thymocytes, however, a cleavable complex by topoisomerase II inhibitors is not essential for the induction of apoptosis, since it was induced by ICRF-154. This suggests that tissue-specific nuclear matrix proteins other than topoisomerase II, including SATP-1 in the thymus, should also be considered. The present findings also suggest that bis(2,6-dioxopiperazine) derivatives are useful agents with which to study the role of topoisomerase II in the regulation of gene expression as well as the role of the nuclear matrix.

4-Piperidinopiperidine-resistant lymphoma cells were resistant to dexamethasone- and A23187-induced apoptosis

4-Piperidinopiperidine is a side residue of CPT-11, a derivative of camptothecin. We have previously established a 4-piperidinopiperidine-resistant lymphoma cell line, 4-pp-R, which was co-resistant to CPT-11. We report here that this cell line is cross-resistant to dexamethasone and A23187 which induce apoptosis in parent RVC cells. Examination of apoptosis-related gene expression by RT-PCR showed that bcl-2 expression was greater in 4-pp-R than in RVC. p53, bax and bcl-xL were expressed at the same level in 4-pp-R and RVC cells. These results suggest that upregulation of bcl-2 in 4-pp-R cells is related to the resistance to CPT-11 as well as to A23187 or dexamethasone.

Synergic stimulation of arabinosylcytosine induced apoptosis in mouse thymocytes by cyclic AMP.

Previous studies demonstrated that arabinosylcytosine (ara-C) induced internucleosomal DNA fragmentation and cell death in mouse thymocytes and that those were inhibited by 1-(5-iso-quinoline-sulfonyl)-2-methylpiperazine hydrochloride, an inhibitor of protein kinases. In the present study, we examined the relationship between the DNA fragmentation induced by ara-C and that by agents which activate intracellular signaling and induce apoptosis in mouse thymocytes. 12-O-tetradecanoyl 13-acetate, a phorbol ester capable of activating protein kinase C or A23187, a calcium ionophore, had no effect on ara-C induced DNA fragmentation. However, ara-C induced DNA fragmentation was synergistically enhanced by cAMP and cAMP receptor agonists. Ara-C inhibited the incorporation of choline into the acid soluble and lipid fractions, and cAMP enhanced this inhibition, suggesting that ara-C metabolites interfere with membrane phospholipid metabolism, partly evoking a certain cellular signaling for apoptosis and interacting with cAMP-evoked signaling.

Preincubation of thymocytes with 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine dihydrochloride (H‐7) induces apoptosis in non‐stimulated thymocytes

1‐(5‐Isoquinolinesulfonyl)‐2‐methylpiperazine hydrochloride (H‐7), an inhibitor of protein kinases, has been shown to inhibit the thymocyte apoptosis induced by various apoptogenic agents. In the present study, when mouse thymocytes were pretreated with H‐7, washed, and cultured for an additional time, apoptosis was induced depending on the preincubation time and the dose of H‐7. The protein kinase C activity in the H‐7‐pretreated and ‐washed cells was not altered, suggesting that an alteration of a certain PKC isoform is related to both the triggering and the progression of apoptosis.

A lymphoma cell line resistant to 4-piperidinopiperidine was less sensitive to CPT-11

Abstract CPT-11 is a promising new anticancer drug in which 4-piperidinopiperidine is a side-chain structure. In the present studies, we examined the role played by 4-piperidinopiperidine in the pharmacological activity of CPT-11. When T-cell lymphoma RVC cells were incubated with 4-piperidinopiperidine at concentrations higher than 50 μg/ml, the cells underwent apoptosis with a nucleosomal ladder of chromosomal DNA on agarose gels in a dose-dependent manner. We then established a cell line resistant to 4-piperidinopiperidine (4-pp-R), which was about 20-fold more resistant to 4-piperidinopiperidine than the parent RVC cells. Moreover, 4-pp-R cells showed coresistance to CPT-11. However, the growth rate and cell cycle population of 4-pp-R cells were not different from those of the parent RVC cells, and there were no differences between the two cell lines with regard to their drug transport system, CPT-11-metabolizing activity, their activity and amount of topoisomerase I, or their sensitivity to either SN-38 or etoposide, suggesting that the cytotoxicity of CPT-11 is not a consequence of the activity of its metabolite SN-38. The present studies suggested that resistance to CPT-11 is in part due to insensitivity to 4-piperidinopiperidine and its metabolites, since 4-piperidinopiperidine was cytotoxic and 4-pp-R cells were less sensitive to CPT-11.