Alternative Splicing Microarrays Reveal Functional Expression of Neuron-specific Regulators in Hodgkin Lymphoma Cells

Abstract Despite the success of combination chemotherapy for the treatment of Hodgkin lymphoma (HL), chemotherapy-resistant disease still remains an unresolved problem with most patients eventually dying due to progression. HL is an ideal disease for targeted therapy. CD30 is a 120-kDa transmembrane glycoprotein belonging to the tumor necrosis factor (TNF)-receptor superfamily and strongly expressed in HL Reed-Sternberg cells. We hypothesize that specific, antibody-mediated, CD30-directed delivery of lentiviral vector constructs, encoding for a lethal cellular toxin, will be an effective anti-lymphoma strategy. However, gene delivery based therapy is limited by the transduction of non-target cells. The technology developed by Morizono et al. [Cell Cycle2005: 4: p854; Nature Medicine2005: 11: p346] demonstrates that lentiviruses can be specifically and effectively directed to target cells by conjugation of an antibody to a modified ZZ SINDBIS viral envelope (m168). In this study, we have used HL cell lines (CD30+: L591, L428, Hs445, RPMI6666) and Burkitt lymphoma (BL) cell lines (CD20+/CD30−: Raji, Ramos). We show that conjugation of an anti-CD30 antibody to m168 (m168anti-CD30), permits specific targeting and transduction of CD30+ Hodgkin lymphoma cells while avoiding CD30− Raji and Ramos cells [range 11–83% for HL cells versus 1–4% for BL cells]. Similarly, targeting of CD20+ cells can be achieved. Several of our HL cell lines (L591, Hs445 and RPMI6666) are CD30+/CD20+ [range 11–25% by flow cytometry] and we show that we can equally transduce these cells with an anti-CD20 antibody conjugated to our lentivirus [range 11–30% for CD20+ HL cells; 3% for L428 cells; 40–47% for BL cells]. In addition, we show in L591 cells, that the re-targeted viruses can transduce a greater percentage of target cells than an unmodified virus [83% for m168anti-CD30 versus 43% for VSVG]. These results demonstrate that the efficacy and specificity of targeted therapy can be greatly enhanced and lay the foundation for the development of more stable anti-CD30 directed lentiviral constructs expressing cellular toxins.

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PU.1-Induced Growth Arrest and Apoptosis in Classical Hodgkin Lymphoma Cells

Blood ◽

10.1182/blood.v118.21.2628.2628 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2628-2628

Author(s):

Hiromichi Yuki ◽

Shikiko Ueno ◽

Hiroaki Niiro ◽

Hiro Tatetsu ◽

Hiroyuki Hata ◽

...

Keyword(s):

Cell Growth ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Conflicts Of Interest ◽

Growth Arrest ◽

Myeloma Cell ◽

G1 Arrest ◽

Classical Hodgkin Lymphoma ◽

Lymphoma Cells ◽

Down Regulation

Abstract Abstract 2628 PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth. In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo. We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

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