Increased expression of protein kinase C alpha plays a key role in retinoic acid-induced melanoma differentiation.

Differentiation of B16 mouse melanoma cells induced by retinoic acid (RA) is preceded by a large increase in protein kinase C alpha (PKC alpha) mRNA and protein. To determine the role of PKC alpha in the differentiation program, we stably transfected B16-F1 cells with a plasmid containing the full length PKC alpha cDNA driven by an SV40 promoter. Two out of thirty-two colonies screened were determined to overexpress PKC by 2-4-fold according to Western blot analysis and PKC enzyme activity. When compared to control cells (wild-type cells and cells transfected only with the neomycin resistance gene), PKC alpha overexpressing clones displayed longer doubling times, diminished anchorage-independent growth, and increased melanin production. RA treatment of control cells mimicked these phenotypic characteristics. When injected subcutaneously into syngeneic mice, PKC alpha overexpressing clones produced smaller tumors and had longer latencies than control cells. These findings, combined with the fact that phorbol esters down-regulate PKC and antagonize RA action suggest that PKC alpha plays a key role in the RA-induced melanoma differentiation.     

Retinoic acid induces expression of SLP-76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.     

Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.     

A MAPK-positive feedback mechanism for BLR1 signaling propels retinoic acid-triggered differentiation and cell cycle arrest

MAPK signaling is required for retinoic acid (RA)-triggered G(0) cell cycle arrest and cell differentiation, but the mechanism is not well defined. In this study, RA is found to cause MAPK activation with sustained association of RAF to MEK or ERK, leading to a MAPK-dependent accumulation of p21(Waf1/Cip1) and binding to CDK2 blocking G(1)/S transition. BLR1, a chemokine receptor, was found to function as a critical component of RA-triggered MAPK signaling. Unlike wild-type parental cells, RA-treated BLR1 knock-out cells failed to show RAF and consequential MEK and ERK phosphorylation, failed to accumulate CDK inhibitors that control G(1)/S transition, and failed to differentiate and arrest in response to RA, whereas ectopically overexpressing BLR1 enhanced MAPK signaling and caused accelerated RA-induced differentiation and arrest. Ectopic overexpression of RAF enhanced BLR1 expression in response to RA, whereas inhibition of RAF or MEK by inhibitors or knockdown of RAF by short interfering RNA diminished RA-induced BLR1 expression and attenuated differentiation and growth arrest. Ectopic expression of the RAF CR3, the catalytically active domain, in the BLR1 knock-out restored RA-induced MAPK activation and the ability to differentiate and arrest, indicating that RAF effects MAPK signaling by BLR1 to propel differentiation/arrest. Taken together, RA induces cell differentiation and growth arrest through activation of a novel MAPK pathway with BLR1 as a critical component in a positive feedback mechanism that may contribute to the prolonged MAPK signaling propelling RA-induced cell cycle arrest and differentiation.     

Retinoids arrest breast cancer cell proliferation: retinoic acid selectively reduces the duration of receptor tyrosine kinase signaling

Retinoic acid (RA) induces cell cycle arrest of hormone-dependent human breast cancer (HBC) cells. Previously, we demonstrated that RA-induced growth arrest of T-47D HBC cells required the activity of the RA-induced protein kinase, protein kinase Calpha (PKCalpha) [J. Cell Physiol. 172 (1997) 306]. Here, we demonstrate that RA treatment of T-47D cells interfered with growth factor signaling to downstream, cytoplasmic and nuclear targets. RA treatment did not inhibit epidermal growth factor (EGF) receptor activation but resulted in rapid inactivation. The lack of sustained EGFR activation was associated with transient rather than sustained association of the EGFR with the Shc adaptor proteins and activation of Erk 1/2 and with compromised induction of expression of immediate early response genes. Inhibiting the activity of PKCalpha, a retinoic acid-induced target gene, prevented the effects of RA on cell proliferation and EGF signaling. Constitutive expression of PKCalpha, in the absence of RA, decreased cell proliferation and decreased EGF signaling. RA treatment increased steady-state levels of the protein tyrosine phosphatase PTP-1C and all measured effects of RA on EGF receptor function were reversed by the tyrosine phosphate inhibitor orthovanadate. These results indicate that RA-induced target genes, particularly PKCalpha, prevent sustained growth factor signaling, uncoupling activated receptor tyrosine kinases and nuclear targets that are required for cell cycle progression.     

Retinoic acid causes MEK-dependent RAF phosphorylation through RARα plus RXR activation in HL-60 cells

Retinoic acid (RA) is known to cause the myeloid differentiation of HL-60 human myeloblastic leukemia cells in a process requiring MEK-dependent ERK2 activation. This RA-induced ERK2 activation appears after approximately 4 h and persists until the cells are differentiated and G0 arrested (Yen et al, 1998). This motivates the question of whether RA also activated RAF as part of a typical RAF/MEK/MAPK cascade. Retinoic acid is shown here to also increase the phosphorylation of RAF, but in an unusual way. Surprisingly, increased RAF phosphorylation is first detectable after 12 to 24 hours by phosphorylation-induced retardation of polyacrylamide gel electrophoretic mobility. The RA-induced increased RAF phosphorylation is still apparent after 72 hours of treatment when most cells are differentiated and G0 arrested. There is a progressive dose-response relationship with 10(-8), 10(-7), and 10(-6) M RA. The RA-induced RAF phosphorylation corresponds to increased in vitro kinase activity. Inhibition of MEK with a PD98059 dose which inhibits ERK2 phosphorylation and subsequent cell differentiation also inhibits RAF phosphorylation. RA-induced MEK-dependent RAF phosphorylation is not due to changes in the amount of cellular MEK. The induced RAF phosphorylation, as well as anteceding ERK2 activation, depends on ligand-induced activation of both an RARalpha receptor and an RXR receptor. This and the slow kinetics of activation suggest a need for prior RA-induced gene expression. In summary, RA induces a MEK-dependent prolonged RAF activation, whose slow onset occurs after ERK2 activation but still well before cell cycle arrest and cell differentiation. The RA-induced increased RAF phosphorylation thus differs from typical mitogenic growth factor signaling, features that may contribute to cell cycle arrest and differentiation instead of division as the cellular outcome.     

B16 mouse melanoma cells selected for resistance to cyclic AMP-mediated growth inhibition are cross-resistant to retinoic acid-induced growth inhibition.

B16 mouse melanoma cells are grown inhibited by cyclic AMP or by retinoic acid (RA). However, the combination of these two agents results in less growth inhibition than either agent alone. In order to investigate this interaction, cells were selected for resistance to 8-bromo-cyclic AMP-induced growth inhibition. Two clones (3 and 7) which demonstrated significant resistance were isolated. When these two clones were treated with retinoic acid (RA) it was observed that they also exhibited different degrees of resistance to this growth inhibitor. This cross-resistance did not appear to be due to a lack of uptake or retention of the respective inhibitors, since the mutants took up and retained more 3H-cAMP and 3H-RA than wild type cells, suggesting that the dual resistance was not due to an amplification of P-glycoprotein. The mutation confering cAMP-resistance did not appear to involve cyclic AMP-dependent protein kinase, since both catalytic activity and the amount of cAMP protein binding was similar in wild type and mutants. Thus, the mutation must be beyond the interaction of cAMP with cAMP-dependent protein kinase. We have previously reported that RA induces protein kinase C in B16 melanoma cells (Niles and Loewy: Cancer Res. 49:4483-4487, 1989). Therefore, we measured the ability of RA to induce protein kinase C in the cyclic AMP-resistant mutants. We found an inverse correlation between RA-induced protein kinase C activity and growth inhibition in these mutants. The data reported here suggest that cyclic AMP regulates some step in the RA signal transduction pathway.     

Control of retinoic acid receptor expression in mouse melanoma cells by cyclic AMP

Retinoic acid receptor (RAR) α and γ mRNAs were constitutively expressed in B16 melanoma cells with or without retinoic acid (RA) treatment. RARβ mRNA, however, was significantly expressed only after exposure to RA. Induction of RARβ by RA occurred within 1 h and was not inhibited by cycloheximide (i.e., did not require new protein synthesis). All three RAR mRNA levels were dramatically decreased with 8-bromo-cyclic AMP treatment and could not be rescued by addition of RA. Analysis of RARγ revealed that this decrease occurred within 1 h of exposure to 8-bromo-cyclic AMP and was not blocked by simultaneous treatment with cycloheximide. The stability of RARγ mRNA was not altered by cyclic AMP treatment. Nuclear extracts from 8-bromo-cyclic AMP-treated cells showed a large decrease in protein binding to a retinoic acid response element (RARE) oligonucleotide compared to control cells. This correlated with a marked reduction of RA-stimulated RARE-reporter gene activity in transfected cells which were treated with cyclic AMP. Pretreatment of B16 cells with cyclic AMP prior to RA addition dramatically reduced induction of PKCα, an early marker of RA-induced cell differentiation. Thus, cyclic AMP can antagonize the action of RA most likely via its ability to inhibit RAR expression. © 1996 Wiley-Liss, Inc.     

Protein kinase Calpha plays a critical role in mannosylerythritol lipid-induced differentiation of melanoma B16 cells

Mannosylerythritol lipid (MEL), a novel extracellular glycolipid from yeast, was found to inhibit the proliferation of mouse melanoma B16 cells in a dose-dependent manner and to induce the apoptosis of B16 cells at concentrations higher than 10 microm (Zhao, X., Wakamatsu, Y., Shibahara, M., Nomura, N., Geltinger, C., Nakahara, T., Murata, T., and Yokoyama, K. K. (1999) Cancer Res. 59, 482-486). We show here that exposure of B16 cells to MEL (5 microm) for 2 days resulted in an increase of the levels of differentiation-associated markers of melanoma cells such as melanogenesis and tyrosinase activity, which were accompanied by morphological changes. The MEL-induced differentiation of B16 cells at this concentration was closely associated with arrest of the cell cycle at G(1) phase, but no significant population of apoptotic cells was identified. Expression of protein kinase Calpha (PKCalpha) was enhanced after exposure of B16 cells to MEL for 48 h. Antisense oligodeoxynucleotides against the mouse gene for PKCalpha prevented MEL-induced melanogenesis in B16 cells. Conversely, the effects of the expression of a constitutively active form of PKCalpha mimicked the effects of MEL on B16 cells. These data suggest that MEL, a yeast-derived glycolipid, triggers the differentiation of B16 melanoma cells through a signaling pathway that involves PKCalpha.     

Retinoic acid increases cyclic AMP-dependent protein kinase activity in murine melanoma cells

The influence of all trans-retinoic acid on cyclic AMP metabolism was examined in B16-F1 mouse melanoma cells. Treatment of these cells with retinoic acid resulted in a dose-dependent inhibition of cell growth which was accompanied by a concentration-dependent increase in both basal and cyclic AMP-stimulated protein kinase activity, Intracellular levels of cyclic AMP, however, were not altered by retinoid treatment. A protein kinase-deficient variant of B16-F1 (MR-4) did not exhibit decreased growth or increased protein kinase activity in response to retinoic acid treatment. At least 24 h of incubation was required before increased protein kinase activity could be detected in treated B16-F1 cells. Retinoic acid treatment increased the Vmax of protein kinase, but the Ka for cyclic AMP activation was not altered. These findings suggest that in B16 mouse melanoma cells, cyclic AMP-dependent protein kinase may be a target for the growth inhibitory effects of the retinoid.