Inhibition of colony formation of NIH 3T3 cells by the expression of the small molecular weight heat shock protein HSP27: involvement of its phosphorylation and aggregation at the C-terminal region.
The ectopic expression of the small molecular weight heat shock protein HSP27 reportedly confers resistance to heat and other types of stress, but our recent findings indicated that it rendered human immortalized fibroblast cells (KMST-6) more sensitive to oxidative stress and caused irreversible growth arrest (Arata et al., 1995, J. Cell. Physiol., 163:458-465). To clarify the relationship between HSP27 and growth regulation, we investigated the effect of overexpression of HSP27 and its mutants on the growth potential of several cell lines. Mammalian expression vectors of the wild-type, hypophosphorylatable, or C-terminal deletion mutants of human HSP27 were constructed from the pRc/CMV plasmid that contained the neomycin-resistant gene. The plasmid was introduced into mouse fibroblasts (NIH 3T3), normal human fibroblasts (TIG-3), Chinese hamster ovary (CHO-K1), or mammary tumor cells (MCF-7), which were then selected in medium containing G418. The number of drug-resistant colonies was significantly decreased by transfection with the expression vector for wild-type HSP27 compared with vector alone, whereas the overexpression of HSP27 in CHO-K1 cells had essentially no effect. The expression vectors of an hypophosphorylatable mutant (pKSm, human HSP27 gene in which codons for Ser-15, -78, and -82 were converted to code for Gly by site-directed mutagenesis) as well as C-terminal deletion mutants in which 12-36 amino acid residues from the C-terminus were deleted had no significant effect on the colony-forming efficiency of NIH 3T3 cells. Cells isolated from G418-resistant colonies formed by transfection of NIH 3T3 cells with the HSP27 expression vector expressed no detectable levels of wild-type HSP27 and did not form stable clonal transformants expressing high levels of HSP27 from NIH 3T3 cells. In contrast, several clones expressing high levels of HSP27 were obtained from CHO-K1 cells transfected with the HSP27 expression vector. In KMST-6 clones expressing high levels of HSP27, the wild-type HSP27 formed aggregates with a mean molecular mass of about 200 kDa as determined by gel filtration, and the size of the oligomers changed with oxidative stress. On the other hand, the size of aggregates of HSP27 encoded by pKSm or C-terminal deletion mutants did not change. These observations indicated that the forced expression of wild-type HSP27 participates in inhibiting the growth of some cell types and that the inhibition may be associated with its phosphorylation and aggregation.
Arata
S
S
Department of Microbiology, Showa University School of Pharmaceutical Science, Tokyo, Japan.
Hamaguchi
S
S
Nose
K
K
D016475:3T3 Cells / Q000166:cytology / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D017353:Gene Deletion; D015870:Gene Expression / Q000502:physiology; D005822:Genetic Vectors / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D010766:Phosphorylation; D010957:Plasmids; D017434:Protein Structure, Tertiary; D014162:Transfection
D006360:Heat-Shock Proteins