Kinetic analysis of Human T-cell Leukemia Virus type 2 expression in chronically-infected cells and patient PBMCs

Introduction: The elucidation of the viral gene expression profile provides useful information in assessing the function of specific viral genes in the process of infection and cellular transformation. HTLV-2 pattern of mRNAs expression produces three major classes of mRNAs: unspliced genomic mRNA for Gag, protease and Pol proteins; singly spliced mRNAs encoding Env and the accessory proteins p28, p22/p20-1 and -2; and a doubly spliced mRNA for the regulatory proteins Tax, Rex and for the p10/p11 and p? accessory ones (Ref.1 and Fig. 1). To date, very little information has been obtained on the temporal regulation of different HTLV-2 transcripts expression in infected cells. Aim of this study was to investigate the kinetics of gene expression from HTLV-2 infected cell lines and from PBMCs of HTLV-2B infected subjects. The expression profile and kinetics of the different transcripts were analysed by real time RT-PCR using splice-junction-specific primers. Results: This approach was used to first determine the steady-state levels of expression for the different viral transcripts in three different cell lines in log phase of growth . Experiments performed indicated that gag/pol is the most abundant transcript. The expression level of env was comparable in the two T-cell lines, Mo-T and C344, infected by the 2A subtype, and was considerably higher than in the B-cells infected with HTLV-2B subtype, where p10/p11 and p? transcripts were below the limit of detection. We next investigated the kinetics of viral transcripts expression in infected BJAB-Gu cells. As in the previous experiment, the absolute copy number of gag/pol was the highest over the time period analysed . Among the accessory transcripts, p28,p22/p20-2 was the most abundant while other regulatory and accessory genes were lower. The analysis of fold variation, reported in g. 4B, indicated that tax/rex and p28, p22/p20-1 showed a biphasic profile with an early peak at 24 hours and a second one at 72 hours, whereas the transcripts gag/pol, env and p28,p22/p20-2 were expressed later.The kinetics of gene expression also was analysed from ex-vivo PBMCs of HTLV-2B infected subjects. Fig. 5 shows a typical pattern of expression. Also in this case, among the mRNAs species, gag/pol was consistently the most abundant transcript, p28, p22/p20-2 was approximately 15 fold lower than gag/pol, followed by tax/rex and p? that were present at approximately 25 fold lower than the unspliced mRNA coding for gag/pol . Very low levels of expression were found for p28, p22/p20-1, while env and p10/p11 transcripts were below the limit of detection. In Fig. 5B the fold variation analysis showed that the first mRNAs expressed were tax/rex and p28,p22/p20-1 with a peak at 4 hours followed by all the other transcripts that showed a later peak at 24 hours.These results indicate that tax/rex is the earliest transcript expressed, while the other genes, coding for accessory and structural proteins, are expressed in a later phase of the viral cycle. Conclusions: The expression of different HTLV-2 genes follows a distinct timing both in infected cell lines and PBMCs isolated from infected patients. The transcript tax/rex is the first to be expressed, thus indicating that it is necessary at the beginning of the infection cycle to transactivate and regulate viral and cellular transcripts. These results also suggest that the control of viral gene expression is highly regulated both in its kinetics and expression level

Therapeutic potential of cladribine in combination with STAT3 inhibitor against multiple myeloma

<p>Abstract</p> <p>Background</p> <p>Cladribine or 2-chlorodeoxyadenosine (2-CDA) is a well-known purine nucleoside analog with particular activity against lymphoproliferative disorders, such as hairy cell leukemia (HCL). Its benefits in multiple myeloma (MM) remain unclear. Here we report the inhibitory effects of cladribine on MM cell lines (U266, RPMI8226, MM1.S), and its therapeutic potential in combination with a specific inhibitor of the signal transducer and activator of transcription 3 (STAT3).</p> <p>Methods</p> <p>MTS-based proliferation assays were used to determine cell viability in response to cladribine. Cell cycle progression was examined by flow cytometry analysis. Cells undergoing apoptosis were evaluated with Annexin V staining and a specific ELISA to quantitatively measure cytoplasmic histone-associated DNA fragments. Western blot analyses were performed to determine the protein expression levels and activation.</p> <p>Results</p> <p>Cladribine inhibited cell proliferation of MM cells in a dose-dependent manner, although the three MM cell lines exhibited a remarkably different responsiveness to cladribine. The IC50 of cladribine for U266, RPMI8226, or MM1.S cells was approximately 2.43, 0.75, or 0.18 μmol/L, respectively. Treatment with cladribine resulted in a significant G1 arrest in U266 and RPMI8226 cells, but only a minor increase in the G1 phase for MM1.S cells. Apoptosis assays with Annexin V-FITC/PI double staining indicated that cladribine induced apoptosis of U266 cells in a dose-dependent manner. Similar results were obtained with an apoptotic-ELISA showing that cladribine dramatically promoted MM1.S and RPMA8226 cells undergoing apoptosis. On the molecular level, cladribine induced PARP cleavage and activation of caspase-8 and caspase-3. Meanwhile, treatment with cladribine led to a remarkable reduction of the phosphorylated STAT3 (P-STAT3), but had little effect on STAT3 protein levels. The combinations of cladribine and a specific STAT3 inhibitor as compared to either agent alone significantly induced apoptosis in all three MM cell lines.</p> <p>Conclusions</p> <p>Cladribine exhibited inhibitory effects on MM cells <it>in vitro</it>. MM1.S is the only cell line showing significant response to the clinically achievable concentrations of cladribine-induced apoptosis and inactivation of STAT3. Our data suggest that MM patients with the features of MM1.S cells may particularly benefit from cladribine monotherapy, whereas cladribine in combination with STAT3 inhibitor exerts a broader therapeutic potential against MM.</p