Induction of 68,000-dalton heat shock proteins by cyclopentenone prostaglandins. Its association with prostaglandin-induced G1 block in cell cycle progression.

K Ohno,M Fukushima,M Fujiwara,S Narumiya

doi:10.1016/s0021-9258(19)77700-x

Abstract

Cyclopentenone prostaglandins (PGs) such as PGA2 and delta 12-PGJ2 act specifically on cells in the G1 phase and induce block of cell cycle progression (Ohno, K., Sakai, T., Fukushima, M., Narumiya, S., and Fujiwara, M. (1988) J. Pharmacol. Exp. Ther. 245, 294-298). In this study, we characterized proteins induced by these PGs in HeLa S3 cells of synchronized growth and examined its association with the cell cycle block. HeLa S3 cells transiently expressed two 68-kDa proteins of isoelectric points of 5.5 and 5.6 in the G1 phase of cell cycle. When G1-enriched cells were incubated with either PGA2 or delta 12-PGJ2, synthesis of these proteins was markedly enhanced. Enhancement by delta 12-PGJ2 was persistent and irreversible, whereas that by PGA2 was reversible. delta 12-PGJ2 also enhanced the synthesis of two additional 68-kDa proteins with isoelectric points of 5.8 and 5.9. On two-dimensional gel electrophoresis, these proteins overlapped exactly with the 68-kDa heat shock proteins induced in cells treated at 43 degrees C for 90 min. They were also indistinguishable from the heat shock proteins in limited proteolysis. When delta 12-PGJ2 was incubated with G2/M phase cells, it induced only a small and transient increase in the 68-kDa proteins. These results suggest that cyclopentenone PGs extensively induce 68-kDa heat shock proteins in the G1 phase HeLa S3 cells and this induction is closely associated with the G1 block of cell cycle progression caused by these PGs.

Highlights

Cyclopentenoneprostaglandins (PGs) such as PGAz nuclear proteins and that this uptake and accumulation are and A"-PGJZ act on cells in the G1 phase closely correlated with the expression of their growth inhibiand induce block of cell cycle progression (Ohno, K., Sakai, T., Fukushima,M., Narumiya, S., and Fujiwara, M. (1988) J
These results suggest that cyclopentenone PGs extensively induce 68-kDa heat shock proteins in the GI phase HeLa S3 cells and this induction is closely associated with the G1 blockof cellcycle modulate the cell cycle progression at a specific point in the GI phase [11,12,13]
Using the same model system, we have analyzed changes in protein synthesis caused by the PGs and examined if such changes were associated witthhe PG-induced GI block of cell cycle progression

Summary

PG or heat shock treatment were washed with PBS andincubated at

Mmol) in 1 mlof methionine-free minimum essential medium containing 10%dialyzed FCS. After reaction, the cells were scraped from culture dishes with a rubber policeman and centrifuged. SDS-polyacrylamide gel electrophoresis of [35S]methionine-labeledproteins (about 0.8-1.0 pCi) were performed in a 5-15% linear gradient gel according to the method of Laemmli [17]. Labeled proteins for two-dimensional gel electrophoresis were extracted according to the method of O'Farrell [18].Protein extracts containing radioactivity of2.5-3pCiwere used in each analysis. Peptide Mapping by Limited Proteolysis-[3SS]Methionine-labeled proteins extracted from HeLa S3 cells were separated by two-dimensional gel electrophoresis. The gel electrophoresis was performed on a15% gel according tothe methods described by Cleveland et al [20].Digested products were detected by fluorography by the use of ENLIGHTNING (Du Pont-New England Nuclear). The cells were washed twice with 2 ml of cold PBSand scraped offby the use of a rubber policeman. Statistical significance of the results was analyzed by the use of Student's t test

RESULTS

Isoelectric point

We next examined the effect of actinomycinD on synthesis

DISCUSSION