Nathan A. Lemp

Splicing mediates the activity of four putative cellular internal ribosome entry sites

A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5′ end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3′ splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3′ splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.

CCAAT/Enhancer-Binding Protein δ: A Molecular Target of 1,25-Dihydroxyvitamin D3 in Androgen-Responsive Prostate Cancer LNCaP Cells

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, inhibits the proliferation of prostate cancer cells. However, the molecular mechanisms by which 1,25(OH)2D3 inhibits the proliferation of these cells remain to be fully elucidated. In this study, we used microarray technology to identify target genes of 1,25(OH)2D3 in androgen-responsive prostate cancer LNCaP cells. 1,25(OH)2D3 up-regulated CCAAT/enhancer-binding protein delta (C/EBPdelta) by approximately 5-fold in these cells. Knockdown of C/EBPdelta expression by RNA interference showed that C/EBPdelta is essential for the significant growth inhibition of LNCaP cells in response to 1,25(OH)2D3 treatment. Moreover, we found that 1,25(OH)2D3 induced C/EBPdelta in other cancer cells, including the estrogen receptor (ER)-expressing MCF-7 and T47D breast cancer cells that are sensitive to the growth inhibitory effects of 1,25(OH)2D3. On the other hand, 1,25(OH)2D3 was not able to induce C/EBPdelta in either androgen receptor-negative PC-3 and DU145 or ER-negative breast cancer MDA-MB-231 cells that were relatively resistant to growth inhibition by 1,25(OH)2D3. Furthermore, forced expression of C/EBPdelta in prostate cancer LNCaP as well as breast cancer MCF-7 and T47D cells dramatically reduced their clonal growth. Taken together, forced expression of C/EBPdelta in cancer cells may be a promising therapeutic strategy.

Prospects for Designing ‘Universal’ Stem Cell Lines

Successful transplantation of conventional tissues between individuals requires matching of human leukocyte associated antigens (HLA), in order to prevent rejection. Although the same principles apply to tissues differentiated from embryonic stem (ES) cells, recent advances in gene delivery and genetic regulation have raised the prospect of engineering grafts with reduced levels of HLA expression. This strategy may mitigate the effects of extensive HLA polymorphism which restricts the availability of suitable donors and necessitates the maintenance of large donor registries. Here, we discuss the potential of employing RNA interference (RNAi) to knockdown HLA expression, enabling allogeneic cells to evade immune recognition. We discuss how lentivirus-mediated delivery of short hairpin RNAs (shRNA) targeting pan-class I and allele-specific HLA achieves efficient, dose-dependent reduction in surface HLA expression in human cells. Thus, by combining genetic engineering and regenerative medicine, RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced and, perhaps even, “universally” compatible cellular grafts.

1016-LBP: RosetteSep HLA – An exceptional method for isolation of lymphocytes from peripheral blood

Aim The isolation of a sufficient quantity of lymphocytes from deceased donor peripheral blood can be problematic. As an OPO HLA lab that serves six different transplant centers, we routinely perform both complement dependent cytotoxicity (CDC) and flow cytometric crossmatching (FCXM) for 15 potential renal recipients plus additional non-renal recipients. Historically, we have used T/B Lympho-Kwik to isolate lymphocytes for FCXM, but this reagent is no longer available. As a replacement method, we adopted a protocol using CD14 and CD15 magnetic beads to negatively select non-lymphocyte cells. However, the lymphocyte yield and purity was frequently insufficient, so we sought to validate a method using RosetteSep HLA Total Lymphocyte Enrichment Cocktail. Methods RosetteSep contains bispecific antibodies which crosslink unwanted cells to RBCs, forming rosettes, which are removed by centrifugation. Buffy coats were pulled from ACD blood tubes and lymphocytes were isolated by centrifugation over IsoPrep and negative selection with CD14/CD15 Dynabeads or RosetteSep. The isolated lymphocytes were counted on a hemacytometer and either used directly for FCXM or further separated by positive selection of B cells using Class II Dynabeads for CDC testing. Results The average cell yield for the RosetteSep isolations was nearly double that of the CD14/CD15 isolations, lymphocyte purity was >95%, and there was an increase in the ratio of B cells to T cells. The viability and CDC reaction of the B cells was comparable between the two methods. The results of the FCXM were also comparable between the two methods, both in terms of T and B cell MFI with negative serum (NS) and T and B cell MCS with patient sera. The increased background on B cells with NS historically seen when using Lympho–Kwik was not observed with RosetteSep. Conclusions Our method using RosetteSep is cost efficient, fast, easy to perform, and significantly increases the yield of lymphocytes compared to CD14/CD15 magnetic bead negative selection.