Protein kinase C is not necessary for transforming growth factor beta-induced growth-arrest in leukemia cell lines

Abstract

Growth and differentiation of blood cell precursors are regulated by cytokines and hormones by mechanisms which are incompletely understood. Protein kinase C (PKC) isozymes are widely regarded as being important in signal transduction pathways. We have shown that one isozyme, PKC β, is uniquely important in mediating phorbol ester-induced growth-arrest in the HL-60 myeloid cell line. 1,25-dihydroxyvitamin D₃ induces differentiation and growth-arrest in many cells. It upregulates the expression of PKC β, potentiating the action of phorbol ester. We tested the hypotheses that cytokines, which arrest the growth of hematopoietic cells, do so by activating PKC β, and that differentiation and growth-arrest induced by 1,25-dihydroxyvitamin D₃ is caused by upregulation of PKC β isozyme gene expression.

The influence on growth of combinations of five cytokines (TNF α, TGF β1, γ-IFN, IL-1, and G-CSF) and 1,25-dihydroxyvitamin D₃ on ten human leukemia cell lines (THP-1, HL-60 S, HL-60 PET, U937, K562, Jurkat, MOLT-4, RPMI 8402, KG-1, and KG-1a) was determined. Four cell lines (THP-1, HL-60 S and PET, and U937) exhibited total growth-arrest when incubated with 1,25-dihydroxyvitamin D₃ followed by TGF β1. The expression by each cell line of mRNA encoding PKC α, β, and δ, both before and after 24 or 48 h of incubation with 1,25-dihydroxyvitamin D₃, was determined. Cell lines sensitive to TGF β1 each expressed PKC δ endogenously, or expression was up-regulated with 1,25-dihydroxyvitamin D₃. U937 cells underexpressed PKC α, and HL-60 PET cells underexpressed PKC β. These data suggested that PKC δ could be responsible for mediating growth-arrest by TGF β1. To test this hypothesis directly, we incubated the cells with two bisindolylmaleimide PKC inhibitors during the addition of 1,25-dihydroxyvitamin D₃ and TGF β1. Surprisingly, the PKC inhibitors did not block the growtharrest induced by 1,25-dihydroxyvitamin D₃ and TGF β1.

This experiment strongly suggests that neither growth-arrest induced by TGF β1 nor the potentiation of this growth-arrest by 1,25-dihydroxyvitamin D₃ is mediated by a PKC isozyme which is inhibitable by the bisindolylmaleimides.