Retinoic acid induces persistent, RAR?-mediated anti-proliferative responses in Epstein-Barr virus-immortalized b lymphoblasts carrying an activated c-myc oncogene but not in Burkitt's lymphoma cell lines

Abstract

We have previously demonstrated that 13‐cis‐retinoic acid (RA), 9‐cis‐RA and all‐trans‐RA (ATRA) powerfully inhibit the proliferation of Epstein‐Barr virus–immortalized B‐lymphoblastoid cell lines (LCLs). The aim of the present study was to assess whether these compounds are effective at inhibiting the growth of B cells at more advanced stages of lymphomagenesis, including fully transformed B lymphocytes. To this end, c‐myc–transfected LCLs (myc‐LCLs) and Burkitt's lymphoma (BL) cell lines were used. We report that 13‐cis‐RA, 9‐cis‐RA and ATRA also markedly inhibit the proliferation of myc‐LCLs by inducing G0/G1 growth arrest as well as enhancing rates of apoptosis. Conversely, all but 1 (DG75) of the 8 BL cell lines investigated were poorly RA‐responsive. Moreover, unlike LCLs and myc‐LCLs, RA‐treated DG75 cells rapidly resumed proliferation upon drug removal. Analysis of cell cycle–regulatory proteins showed that, as in LCLs, strong up‐regulation of p27Kip−1 and increased levels of under‐phosphorylated pRb and p130 were detected in RA‐treated DG75 cells. While the catalytic activity of all 3 G1‐associated CDKs (CDK2, CDK4 and CDK6) was strongly inhibited in RA‐treated LCLs, only CDK2‐associated kinase activity was reduced in DG75 cells arrested in G0/G1 by RA. Moreover, RA‐treated DG75 cells failed to show the down‐regulation of cyclin D3 observed in LCLs. Use of receptor‐selective agonists and antagonists showed that in LCLs and RA‐responsive BL cells, RA‐induced growth arrest is mainly mediated by RARα. The RARα‐selective agonist Ro 40‐6055 was also effective at very low concentrations (10–10 M). Nevertheless, comparable levels of RARα mRNA were found in RA‐responsive and ‐resistant BL cell lines, indicating that mechanisms different from transcriptional deregulation of RARα probably underlie the differential responsiveness of BL cells. Int. J. Cancer 86:375–384, 2000. © 2000 Wiley‐Liss, Inc.

Inhibition of oxidative phosphorylation underlies the antiproliferative and proapoptotic effects of mofarotene (Ro 40-8757) in Burkitt's lymphoma cells

“…Similar effects were also induced in LCLs carrying an activated c-myc oncogene (Cariati et al, 2000). In contrast, RA exerted significant antiproliferative responses only in a minority of BL cell lines, inducing a growth arrest that was fully reversible upon drug removal (Cariati et al, 2000). In the search for retinoids active against BL, we found that the arotinoid mofarotene (Ro 40-8757) was able to markedly inhibit the growth of BL cells but not of EBV-immortalized LCLs.…”

“…In particular, we have shown that 13-cis-RA, 9-cis-RA, and ATRA exert a strong and persistent antiproliferative effect on EBV-immortalized LCLs at concentrations corresponding to therapeutically achievable plasma levels (Pomponi et al, 1996). Similar effects were also induced in LCLs carrying an activated c-myc oncogene (Cariati et al, 2000). In contrast, RA exerted significant antiproliferative responses only in a minority of BL cell lines, inducing a growth arrest that was fully reversible upon drug removal (Cariati et al, 2000).…”

Retinoic acid stabilizes p27Kip1 in EBV-immortalized lymphoblastoid B cell lines through enhanced proteasome-dependent degradation of the p45Skp2 and Cks1 proteins

“…In these cells, RA induces a marked upregulation of p27 Kip1 , leading to inhibition of CDK2, CDK4, CDK6 kinase activity and ultimately resulting in reduced pRb phosphorylation and G 0 /G 1 growth arrest (Pomponi et al, 1996;Zancai et al, 1998). Similar findings were also observed in LCLs carrying a constitutively activated c-myc oncogene, indicating that RA is effective in inhibiting the proliferation also of immortalized B cells that have undergone a further step towards the malignant phenotype (Cariati et al, 2000). On these grounds, and considering the central role of p27 Kip1 in mediating the antiproliferative activity exerted by RA on EBV-immortalized and neoplastic B lymphocytes, we have carried out a study aimed at defining the mechanisms by which RA upregulates p27 Kip1 in the B cell lineage.…”

“…We have previously demonstrated that the antiproliferative effect exerted by RA isomers in immortalized and neoplastic B lymphocytes is associated with the ability of these compounds to induce p27 Kip1 protein accumulation, which results in G0/G1 arrest (Zancai et al, 1998;Cariati et al, 2000). The results of the present study indicate that, in LCLs, the ubiquitinproteasome pathway is involved in regulating the turnover of p27 Kip1 and that RA increases p27 Kip1 stability by post-translational mechanisms, resulting in a decreased proteasome-dependent degradation of the protein.…”

“…To this end, we set up a system to induce adoptive expression of p45 Skp2 in B cells using the retroviral expression plasmid pBabe Puro. Considering that in LCLs we repeatedly verified a very low efficiency of infection, we induced ectopic p45 Skp2 expression in the DG75 Burkitt lymphoma B cell line in which RA recapitulates all the cellular and biochemical events observed in LCLs, including the G0-G1 cell cycle arrest and p27 Kip1 upregulation (Cariati et al, 2000). As shown in Figure 5a, DG75 cells infected with the pBabe-Skp2 vector showed a strong upregulation of p45 Skp2 protein levels and this correlated with a decreased p27 Kip1 expression.…”