Silvana Rizzo

Distinct functional significance of Akt and mTOR constitutive activation in mantle cell lymphoma

Functional characterization of signaling pathways that critically control mantle cell lymphoma (MCL) cell growth and survival is relevant to designing new therapies for this lymphoma. We herein demonstrate that the constitutive activation of Akt correlates with the expression of the phosphorylated, inactive form of PTEN. Phosphatidyl-inositol-3 kinase (PI3-K)/Akt or mammalian target of rapamycin (mTOR) inhibition decreased the growth of both primary MCL cultures and established cell lines and antagonizes the growth-promoting activity of CD40 triggering and IL-4. These effects are mediated by nuclear accumulation of the p27(Kip1) inhibitor induced by down-regulation of the p45(Skp2) and Cks1 proteins, which target p27(Kip1) for degradation. Moreover, Akt inhibition down-regulated cyclin D1 by promoting its proteasome-dependent degradation driven by GSK-3. Intriguingly, mTOR inhibition affected cyclin D1 proteolysis only in MCL cells in which GSK-3 is under the direct control of mTOR, suggesting that different MCL subsets could be differently responsive to mTOR inhibition. Finally, PI3-K/Akt inhibitors, but not rapamycin, induced variable levels of caspase-dependent apoptosis and reduced telomerase activity. These results indicate that Akt and mTOR activation have distinct functional relevance in MCL and suggest that targeting Akt may result in more effective therapeutic effects compared with mTOR inhibition.

Latent membrane protein 1 of Epstein-Barr virus activates the hTERT promoter and enhances telomerase activity in B lymphocytes

ABSTRACT Transformation of primary B lymphocytes by Epstein-Barr virus requires the establishment of a strictly latent infection, the expression of several latent viral proteins, and sustained telomerase activity. Our previous findings indicated that induction of hTERT, the rate-limiting catalytic unit of the telomerase complex, was associated with the expression of the viral latent membrane protein 1 (LMP1). In the present study, we demonstrate that ectopic expression of LMP1 in BJAB and Ramos B cells resulted in an increase of hTERT transcripts, thus suggesting that LMP1 acts at the transcriptional level. This was confirmed by transient expression of a luciferase reporter plasmid containing the hTERT promoter cotransfected with an LMP1-expressing vector or transfected into B cells in which LMP1 expression was inducible. Consistently, silencing of LMP1 by small interfering RNA resulted in a reduction of hTERT transcripts. We also provide evidence indicating that LMP1-induced hTERT activation is independently mediated by NF-κB and by mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways, whereas CD40, Akt, and mTOR signaling has no involvement. Moreover, our results do not support a role for c-Myc in mediating these effects on hTERT, since ectopic expression of LMP1 did not upregulate c-Myc and silencing of this oncogene or E box mutagenesis failed to inhibit LMP1-induced hTERT activation. These findings indicate that LMP1 simultaneously modulates multiple signal transduction pathways in B cells to transactivate the hTERT promoter and enhance telomerase activity, thus confirming the pleiotropic nature of this viral oncoprotein.

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 TGFβ 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 TGFβ. 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.

Retinoic acid induces persistent, RARα-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 Burkitts 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.

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

Retinoic acid (RA) arrests the growth of EBV-immortalized lymphoblastoid B cell lines (LCLs) by upregulating the cyclin-dependent kinase inhibitor p27Kip1. Here, we show that in LCLs, RA inhibits ubiquitination and proteasome-dependent degradation of p27Kip1, a phenomenon that is associated with downregulation of Thr187 phosphorylation of the protein, whereas the phosphorylation on Ser10 is unaffected. Furthermore, we demonstrate that RA downregulates the expression of the p45Skp2 and Cks1 proteins, two essential components of the SCFSkp2 ubiquitin ligase complex that target p27Kip1 for degradation. Downregulation of p45Skp2 and Cks1 occurs before the onset of growth arrest and is due to enhanced proteasome-mediated proteolysis of these proteins. Moreover, overexpression of p45Skp2 in DG75 cells prevents p27Kip1 protein accumulation and promotes resistance to the antiproliferative effects of RA. Treatment with Leptomycin B (LMB) blocked the translocation of p27Kip1 to the cytoplasm and prevented its degradation, indicating that CRM1-dependent nuclear export is required for p27Kip1 degradation. The shuttle protein p38Jab1, however, does not accumulate in the nucleus upon LMB treatment, nor does it interact with p27Kip1. Conversely, p45Skp2 is associated with p27Kip1 both in the nucleus and in the cytoplasm, accumulating within the nuclei after exposure to LMB and co-localizing with the exportin CRM1, suggesting a possible involvement of p45Skp2 in CRM1-dependent nuclear export of p27Kip1. These results indicate that downregulation of p45Skp2 is a key element underlying RA-induced p27Kip1 stabilization in B cells, resulting in an impaired targeting of the protein to the ubiquitin–proteasome pathway and probably contributing to the nuclear accumulation of p27Kip1.

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 Hodgkins 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. Int. J. Cancer80:240–249, 1999.

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

In the search for retinoids active against Burkitts lymphoma (BL), we found that the arotinoid mofarotene (Ro 40-8757) induced strong antiproliferative and apoptotic responses in most established BL cell lines as well as in primary BL cells. Ro 40-8757-induced apoptosis is associated with mitochondrial membrane depolarization, activation of caspase-3 and -9, and enhanced production of reactive oxygen species. These effects were related to a transient drop in intracellular ATP content, probably favored by a downregulation of NADH dehydrogenase subunit-1, a component of the mitochondrial respiratory chain (MRC) Complex I. Inhibition of MRC with thenoyltrifluoroacetone suppressed both the ATP recovery and apoptosis, confirming that the effects of Ro 40-8757 are mediated by changes in mitochondrial function. Compared to EBV-negative lines, EBV-carrying BLs were more resistant to Ro 40-8757-induced apoptosis. EBV infection and ectopic LMP-1 expression increased the resistance of BL cells to Ro 40-8757-induced apoptosis, probably through bcl-2 upregulation. Finally, we also show that 2-methoxyoestradiol, an inhibitor of the scavenger enzymes superoxide dismutases, enhanced Ro 40-8757-mediated apoptosis. These findings provide the rationale for evaluating the clinical efficacy of Ro 40-8757 in BL patients and suggest that the combination of Ro 40-8757 with inhibitors of scavenger enzymes may be a promising therapeutic approach for this aggressive lymphoma.

A coordinated proto-oncogene expression characterizes MCF 247 murine leukemia virus-induced T-cell lymphomas irrespectively of proviral insertion affecting myc loci

Proto-oncogene transcriptional activation was analyzed in a group of MCF 247 MuLv-induced T-cell lymphomas to identify transformation-specific gene activations and determine whether the proviral insertion near a myc gene could promote a peculiar mechanism of transformation through a differential proto-oncogene expression pattern. Of the six lymphomas analyzed, three showed the MCF 247 provirus integrated within the N-myc locus, one carried the provirus integrated near c-myc, whereas for the remaining two, no evidence of proviral integrations in any of the known myc loci was obtained. Independently of the integrative events, the pattern of proto-oncogene expression was almost identical in all six lymphomas. These findings seem to rule out the existence of a peculiar pathway of transformation associated with the proviral insertion near a myc locus. Moreover, the transcription pattern observed was qualitatively identical to that displayed by normal thymocytes; only quantitative differences in c- or N-myc, c-myb and Ha-ras were observed. These results suggest that the T-cell proto-oncogene activation program is not qualitatively affected by the transforming event(s).

SPONTANEOUS MUTATION OF CELL ONCOGENES PLAYS A MINOR ROLE IN NEOPLASTIC TRANSFORMATION OF VIRUS-INDUCED MURINE T-CELL LYMPHOMAS

Mink cell focus-forming viruses (MCF) are slow-transforming retroviruses that are able to accelerate the appearance of T-cell lymphomas when injected in newborn AKR mice. Activation of proto-oncogenes by proviral insertion is thought to be the major mechanism by which these viruses exert their oncogenic potential. However, molecular phenomena not strictly virus-determined, such as mutations in cellular oncogenes/tumor suppressor genes or chromosome aberrations, have been hypothesized to contribute to the achievement of the fully neoplastic phenotype in MCF-infected mice. To evaluate the role of spontaneous mutagenesis phenomena in murine virus-induced lymphomagenesis, we analyzed a series of 18 MCF247-induced thymic lymphomas and derived cell lines for the presence of p53 and c-ras gene mutations. Only 1 mutation at the p53 gene and 1 mutation at the ki-ras gene were detected in our study. Our results suggest that spontaneous mutagenesis plays a minor role in virus-induced lymphomagenesis and support the notion that multiple proviral insertions could be the prevalent mechanism of transformation in this experimental system.