Interferons and interleukin 6 suppress phosphorylation of the retinoblastoma protein in growth-sensitive hematopoietic cells.

Abstract

One approach to identify postreceptor molecular events that transduce the negative-growth signals of inhibitory cytokines is to analyze the cytokine-induced modifications in the expression of cell-cycle-controlling genes. Here we report that suppression of phosphorylation of the retinoblastoma gene product (pRb) is a receptor-generated event triggered by interferons and interleukin 6 (IL-6) in hematopoietic cell lines. The conversion of pRb to the underphosphorylated forms occurs concomitantly with the decline in c-myc protein expression and both events precede the G./Gj-phase arrest induced by the cytokines. Loss of IL-6-induced c-myc responses in cells that have been stably transfected with constitutive versions of the c-myc gene abrogates the typical GO/G1-phase arrest but does not prevent the specific dephosphorylation of pRb. Conversely, depletion of protein kinase C from cells interferes with part of the interferon-induced suppression of pRb phosphorylation and relieves the G./Gj-phase cell-cycle block without affecting the extent of c-myc inhibition. None of the cytokines, including transforming growth factor , reduce the phosphorylation of pRb in S-phase-blocked cells. In contrast, the other IL-6-induced molecular responses, including the decline in c-myc mRNA levels, are not phase-specific and develop normally in S-phase-blocked cells that are depleted of the underphosphorylated functional forms of pRb. These experiments distinguish between the reduction ofc-myc expression and the suppression of pRb phosphorylation, which occur independently of each other, and suggest that the development of the interferon-or IL-6-induced GO/Gl-specific arrest requires at least these two receptor-generated events.

Interleukin-6 dependent induction of the cyclin dependent kinase inhibitor p21WAF1/CIP1 is lost during progression of human malignant melanoma

“…Consistent with this ®nding, an increased level of p21 WAF1/CIP1 along with accumulation of hypophosphorylated pRb protein, have been detected in M1 leukemia cells induced to di erentiate by IL-6 treatment (Resnitzky et al, 1992). Furthermore, treatment of B16 ± F10.9 mouse metastatic melanoma cells with IL-6 in the presence of exogenously added soluble IL-6R alpha subunit (gp 80) was shown recently to induce growth arrest, differentiation and expression of p21 WAF1/CIP1 (Oh et al, 1997).…”

Interferon-induces expression of cyclin-dependent kinase-inhibitors p21WAF1 and p27Kip1 that prevent activation of cyclin-dependent kinase by CDK-activating kinase (CAK)

“…To understand the mechanism of IFN-mediated growth-inhibition of cells in the G0/G1-phase of the cell-cycle, we and others have earlier shown that IFNa-treatment of Daudi cells leads to enhanced expression of under-phosphorylated form of pRB (Kumar and Atlas, 1992;Resnitzky et al, 1992). To illustrate this e ect, Figure 1 shows the kinetics of inhibition of pRB phosphorylation ( Figure 1a) and inhibition of CDK-2 associated H-1 kinase activity without any e ect on CDK-2 expression (Figure 1b).…”

“…To understand the mechanism of inhibitory e ect of IFN in the G0/G1-phase of the cell cycle, we and others have earlier shown that IFN-a enhances the expression of underphosphorylated RB protein (pRB) by inhibiting the pRB phosphorylation in human Burkitt's lymphoma Daudi cells (Burke et al, 1992;Kumar and Atlas, 1992;Resnitzky et al, 1992). We have also demonstrated that IFN-g inhibits the growth of human epidermoid A-431 cells in the G0/G1-phase of the cell cycle (Kumar and Mendelsohn, 1989).…”

Transcriptional repression of the E2F-1 gene by interferon-α is mediated through induction of E2F-4/pRB and E2F-4/p130 complexes

“…Regarding the mechanisms of growth suppression, IFNs have been reported to induce down-regulation of c-myc (Einat et al, 1985), dephosphorylation of the retinoblastoma susceptibility gene product (pRB) (Resnitzky et al, 1992), inhibition of both cyclins and cyclin-dependent kinases (CDK) in their expression and activities (Thomas, 1988;Yamada et al, 1994), induction of CDK inhibitors (Chin et al, 1996;Sangfelt et al, 1997) and modulation of E2F activity (Melamed et al, 1993). IFNs can also suppress the signaling pathway of growth factors such as plateletderived growth factor and interleukin-6, which may contribute to growth inhibition of factor-dependent cells (Xu et al, 1994).…”