Damaged microtubules can inactivate BCL-2 by means of the mTOR kinase.

Rapamycin, a specific inhibitor of the serine/threonine mTOR kinase, markedly inhibited both cell growth and apoptosis in human B-cell lines. Besides arresting cells in G(1) by increasing p27(kip1), rapamycin tripled the cellular level of the BCL-2 protein. The activity was dose-dependent and specific for the p27(kip1) and BCL-2 proteins. Rapamycin did not affect bcl-2 mRNA although it increased cellular BCL-2 concentration by inhibiting phosphorylation, a mechanism initiating the decay process. To add new insight, we combined rapamycin treatment with treatment by taxol, which, by damaging microtubules, can phosphorylate BCL-2 and activate apoptosis. It was found that the mTOR kinase was activated in cells treated with taxol or with nocodazole although it was inhibited in cells pre-treated with rapamycin. BCL-2 phosphorylation, apoptosis and hyperdiploidy were also inhibited by rapamycin. In contrast, taxol-induced microtubule stabilization or metaphase synchronization were not inhibited by rapamycin. Taken together, these findings indicate that mTOR belongs to the enzymatic cascade that, starting from damaged microtubules, phosphorylates BCL-2. By regulating apoptosis, in addition to the control of a multitude of growth-related pathways, mTOR plays a nodal role in signaling G(1) and G(2)-M events.

Glucocorticoids promote the proliferation and antagonize the retinoic acid-mediated growth suppression of Epstein-Barr virus-immortalized B lymphocytes.

Glucocorticoids are able to release Epstein-Barr virus-immortalized (EBV-immortalized) lymphoblastoid B cell lines (LCLs) from the persistent growth arrest induced in these cells by retinoic acid (RA). Moreover, physiologic concentrations of glucocorticoids efficiently antagonized LCL growth inhibition induced by 13-cis-RA; 9-cis-RA; all-trans-RA; and Ro 40-6055, an RA alpha receptor (RAR alpha) selective agonist. RAR alpha expression levels, however, were not affected by glucocorticoids. Glucocorticoids, but not other steroid hormones, directly promote LCL proliferation, a phenomenon that was mainly mediated by down-regulation of the cyclin-dependent kinase (CDK) inhibitor p27(Kip-1). Moreover, glucocorticoids contrasted the up-regulation of p27(Kip-1), which was underlying the RA-induced LCL growth arrest, thereby indicating that glucocorticoids and RA signalings probably converge on p27(Kip-1). Both antagonism of RA-mediated growth inhibition and promotion of LCL proliferation were efficiently reversed by the glucocorticoid receptor (GR) antagonist RU486, indicating that all of these effects were mediated by GR. Of note, RU486 also proved to be effective in vivo and, in mice, was able to significantly inhibit the growth of untreated LCLs as well as LCLs growth-arrested by RA in vitro. These findings provide a rational background to further evaluate the possible role of glucocorticoids in the pathogenesis of EBV-related lymphoproliferations of immunosuppressed patients. Moreover, GR antagonists deserve further consideration for their possible efficacy in the management of these disorders, and the use of schedules, including both RA and a GR antagonist, may allow a more thorough evaluation of the therapeutic potential of RA in this setting. (Blood. 2000;96:711-718)

Retinoic acid induces persistent, RARalpha-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.

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 G(0)/G(1) 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 p27(Kip-1) and increased levels of under-phosphorylated pRb and p130 were detected in RA-treated DG75 cells. While the catalytic activity of all 3 G(1)-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 G(0)/G(1) 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 RARalpha. The RARalpha-selective agonist Ro 40-6055 was also effective at very low concentrations (10(-10) M). Nevertheless, comparable levels of RARalpha mRNA were found in RA-responsive and -resistant BL cell lines, indicating that mechanisms different from transcriptional deregulation of RARalpha probably underlie the differential responsiveness of BL cells.

Biologically relevant phenotypic changes and enhanced growth properties induced in B lymphocytes by an EBV strain derived from a histologically aggressive Hodgkin's disease.

Epstein-Barr virus (EBV) isolates show a wide genomic heterogeneity, and a key issue is whether distinct strain variations may contribute to the development and/or malignancy of EBV-related disorders. Herein, we report on the virologic and biologic characterization of an EBV strain derived from a cyto-histologically aggressive EBV-related Hodgkin's disease (HD) (case HD-3) showing a high number of "anaplastic" Reed-Sternberg cells expressing markedly high levels of CD30, CD40 and LMP-1. The HD-3-derived EBV showed strong in vitro immortalizing properties, as suggested by the unusually high number of spontaneous lymphoblastoid cell lines (LCLs) obtained from the patient. Immunofluorescence and immuno-cytochemical analyses showed that HD-3 LCLs expressed significantly higher levels of CD23, CD30, CD38, CD39, CD40 and CD71 antigens and CD54 and CD58 adhesion molecules than B95.8 LCLs. In contrast, the expression of CD11a, CD24, CD95, bcl-2, LMP-1 and EBNA-2 was similar in both groups of LCLs. These phenotypic changes are consistent with the induction of a pronounced activation status and are not dependent on the cellular background, having been closely reproduced by the same virus in LCLs from an unrelated donor (DEN-HD-3 LCLs). HD-3 LCLs were able to grow in vitro at low serum concentrations (up to 0.1%) and were significantly more clonogenic in soft agarose than B95.8 LCLs. Moreover, although no evidence of tumor formation was observed in nude mice injected with B95.8 LCLs, all 5 spontaneous LCLs of patient HD-3 and the 2 DEN-HD-3 LCLs grew in transplanted animals as lymphoproliferations composed of EBER+, LMP-1+ cells. Our findings indicate that the biologic properties of the HD-3 EBV strain are significantly different from those of the B95.8 virus and may have contributed to the cytologic and histo-pathologic malignancy of this HD case. Moreover, molecular characterization of the HD-3 EBV genome identified a 63-bp deletion within the 3' end of the LMP-1 gene as a likely significant change that may be responsible, at least in part, for the biologically relevant phenotypic modifications and enhanced in vitro and in vivo growth potential induced in B lymphocytes by this virus strain.

Retinoic acid-mediated growth arrest of EBV-immortalized B lymphocytes is associated with multiple changes in G1 regulatory proteins: p27Kip1 up-regulation is a relevant early event.

EBV-immortalized lymphoblastoid B cell lines (LCLs) are a suitable in vitro model for the study of EBV-related lymphoproliferative disorders of immunosuppressed patients. We have previously shown that 9-cis-, 13-cis- and all-trans-retinoic acid (RA) powerfully inhibit LCL proliferation at concentrations corresponding to therapeutically achievable plasma levels (10(-6) M). Herein we show that RA-induced LCL accumulation in the G0/G1 phases correlated with the loss of the catalytic activity of all three G1-associated CDKs (CDK2, CDK4 and CDK6) and with increased levels of underphosphorylated pRb and, in some LCLs, p130. LCLs arrested in G0/G1 by RA also showed a significant decrease in the protein levels of cyclins D2, D3 and A, together with a reduction in the amount of cyclin D associated with CDK4 and CDK6, probably accounting for the inhibition of the relative kinase activity. In addition, RA-treated LCLs showed a marked up-regulation of the CDK inhibitor (CKI) p27Kip-1 at the protein but not mRNA level, which correlated with a progressive increase of p27Kip-1 in CDK2 complexes (more than 2.5-fold) and with a reduction in the active phosphorylated form of CDK2. p27Kip-1 may also contribute to the inhibition of CDK4 kinase activity, as the amount of CDK4-associated p27Kip-1 was increased by 50% after RA exposure. p27Kip-1 up-regulation stably persisted for more than one week after RA withdrawal concomitantly with the maintenance of the proliferative block. Moreover, neutralization of TGFbeta did not affect the growth inhibitory activity of RA, suggesting that LCL growth arrest induced by these retinoids is probably not mediated by a pathway directly involving TGFbeta. Overall, these results demonstrate that RA treatment of EBV-immortalized B lymphocytes is associated with multiple effects on G1 regulatory proteins, including p27Kip1 up-regulation, decreased levels of cyclins D2, D3 and A, and inhibition of CDK2, CDK4 and CDK6 activity, which ultimately result in reduced pRb phosphorylation and G0/G1 growth arrest.

In vitro effects of retinoids on the proliferation and differentiation features of Epstein-Barr virus-immortalized B lymphocytes.

Retinoids have been shown to be effective in the chemoprevention and treatment of certain human malignancies. In this review, we will summarize our recent results concerning the effects of retinoids on the proliferation and differentiation of Epstein-Barr virus (EBV)-immortalized lymphoblastoid B-cell lines (LCLs), an in vitro model of EBV-related lymphoproliferative disorders arising in immunosuppressed hosts. Retinoids proved to be powerful inhibitors of the proliferation of EBV-infected LCLs in vitro, with 13-cis-retinoic acid (RA), all-trans-RA, and 9-cis-RA being the most effective compounds. Of note, retinoid-induced growth arrest in vitro appears irreversible at drug concentrations (10(-6) mol/L) which may be reached in man after oral systemic therapy. The antiproliferative activity exerted by retinoids on LCLs is a generalized phenomenon usually associated with a progressive accumulation in G0/G1 phases of treated cells. The strong upregulation of p27Kip1 invariably observed in cells exposed to retinoids may contribute to the decreased number of cycling cells, probably by inhibiting the transition from the G1 to S phase. Moreover, we obtained evidence indicating that the antiproliferative effects of retinoids are not dependent on the induction of terminal differentiation of EBV-immortalized B lymphocytes. In fact, the modifications induced by retinoids relative to LCL morphology, phenotype (downregulation of CD19, HLA-DR, and s-Ig, and upregulation of CD38 and c-Ig), and IgM production were highly variable among the lines tested and often only slightly relevant. Finally, the antiproliferative activity exerted by retinoids on LCLs is not mediated by a direct modulation of viral latent antigens, since EBNA-2 and LMP- downregulation was a late event detected only in some cell lines. These results indicate that retinoids may be useful in the medical treatment of EBV-related lymphoproliferative disorders of immunosuppressed patients, particularly in the earlier phases of these diseases.

Epstein-Barr virus strains with latent membrane protein-1 deletions: prevalence in the Italian population and high association with human immunodeficiency virus-related Hodgkin's disease.

Six Epstein-Barr virus (EBV)-related lymphoproliferative disorders were investigated to verify whether the EBV strain harbored by neoplastic cells had the same EBNA-2 and latent membrane protein-1 (LMP-1) DNA sequences of the virus carried by normal lymphocytes of the same patients. Within each case, the analysis of neoplastic lymph nodes, reactive lymphadenopathies, and/or EBV+ spontaneous lymphoblastoid cell lines gave concordant results with respect to type-specific EBNA-2 region and LMP-1 gene. In particular, five cases showed the same deletion in the 3' end of the LMP-1 gene in both normal and neoplastic cells. We also determined the prevalence of LMP-1 deletions in a large series of normal peripheral blood mononucleated cells (PBMCs) from Italian individuals. The analysis showed that 50% (9 of 18) of PBMCs from human immunodeficiency virus (HIV)-seronegative donors carried a 30-bp deletion in the C-terminal portion of the LMP-1 gene, whereas a nondeleted fragment was amplified in about 44% (8 of 18) of the cases. Only one sample (5.6%) showed the amplification of a full-length LMP-1 band together with a deleted fragment. Similarly, PBMCs from HIV-infected patients showed an almost equivalent prevalence of full-length (17 of 37, 46%) and deleted (16 of 37, 43.2%) LMP-1 fragments, whereas about 11% of samples (4 of 37) showed evidence of double infections. Of note, deletions in the LMP-1 gene were detected with similar prevalence values in EBV+ Hodgkin's disease (HD) (13 of 30, 43.3%) and non-Hodgkin's lymphoma (NHL) (2 of 5, 40%) cases from HIV-seronegative patients and in HIV-related, EBV+ NHLs (4 of 7, 57.1%). Conversely, a 30-bp LMP-1 deletion was found in 10 of 12 HIV-associated HD cases (83%), a prevalence significantly higher than that detected in HIV-unrelated HD (P = .01). These findings indicate that: (1) the same EBV strain carrying LMP-1 deletions is harbored by normal and neoplastic cells of patients with EBV+ disorders, ruling out that these mutations might result from immunoselection phenomena; (2) in the Italian population, the prevalence of LMP-1 deletion mutants is comparable to that of EBV strains with full-length LMP-1; (3) HIV-induced immunosuppression is not associated with an increased prevalence of LMP-1 deletions in PBMCs; and (4) HIV-related HD cases, but not those of HIV-seronegative Italian patients, are closely correlated with the presence of LMP-1 deletions, suggesting that infection with these strains may increase the risk of developing HD in the HIV setting.

Retinoids irreversibly inhibit in vitro growth of Epstein-Barr virus-immortalized B lymphocytes.

Natural and synthetic retinoids have proved to be effective in the treatment and prevention of various human cancers. In the present study, we investigated the effect of retinoids on Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines (LCLs), since these cells closely resemble those that give rise to EBV-related lymphoproliferative disorders in the immunosuppressed host. All six compounds tested inhibited LCL proliferation with no significant direct cytotoxicity, but 9-cis-retinoic acid (RA), 13-cis-RA, and all-trans-RA (ATRA) were markedly more efficacious than Ro40-8757, Ro13-6298, and etretinate. The antiproliferative action of the three most effective compounds was confirmed in a large panel of LCLs, thus appearing as a generalized phenomenon in these cells. LCL growth was irreversibly inhibited even after 2 days of treatment at drug concentrations corresponding to therapeutically achievable plasma levels. Retinoid-treated cells showed a marked downregulation of CD71 and a decreased S-phase compartment with a parallel accumulation in Gzero/ G1 phases. These cell cycle perturbations were associated with the upregulation of p27 Kip1, a nuclear protein that controls entrance and progression through the cell cycle by inhibiting several cyclin/cyclin-dependent kinase complexes. Unlike what is observed in other systems, the antiproliferative effect exerted by retinoids on LCLs was not due to the acquisition of a terminally differentiated status. In fact, retinoid-induced modifications of cell morphology, phenotype (downregulation of CD19, HLA-DR, and s-Ig, and increased expression of CD38 and c-Ig), and IgM production were late events, highly heterogeneous, and often slightly relevant, being therefore only partially indicative of a drug-related differentiative process. Moreover, EBV-encoded EBV nuclear antigen-2 and latent membrane protein-1 proteins were inconstantly downregulated by retinoids, indicating that their growth-inhibitory effect is not mediated by a direct modulation of viral latent antigen expression. The strong antiproliferative activity exerted by retinoids in our experimental model indicates that these compounds may represent a useful tool in the medical management of EBV-related lymphoproliferative disorders of immunosuppressed patients.