Lentiviral vectors (LVs) offer several advantages over traditional oncoretroviral vectors. LVs efficiently transduce slowly dividing cells, including hematopoietic stem-progenitor cells (HSCs), resulting in stable gene transfer and expression. Additionally, recently developed self-inactivating (SIN) LVs allow promoter-specific transgene expression. For many gene transfer applications, transduction of more than one gene is needed. We obtained inconsistent results in our attempts to coexpress two transgenes linked by an internal ribosomal entry site (IRES) element in a single bicistronic LV transcript. In more than six bicistronic LVs we constructed containing a gene of interest followed by an IRES and the GFP reporter gene, GFP fluorescence was undetectable in transduced cells. We therefore investigated how to achieve consistent and efficient coexpression of two transgenes by LVs. In a SIN LV containing the elongation factor 1alpha promoter, we included a second promoter from cytomegalovirus, the phosphoglycerate kinase gene, or the HLA-DRalpha gene. Using a single LV containing two constitutive promoters, we achieved strong and sustained expression of both transgenes in transduced engrafting CD34(+) HSCs and their progeny, as well as in other human cell types. Thus, such dual-promoter LVs can coexpress multiple transgenes efficiently in a single target cell and will enable many gene transfer applications.

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C/EBP␣ is required for generation of granulocyte-monocyte progenitors, but the subsequent role of C/EBP␣ in myeloid lineage commitment remains uncertain. We transduced murine marrow cells with C/EBP␣-estradiol receptor (ER) or empty vector and subjected these to lineage depletion just prior to culture in estradiol with myeloid cytokines. This protocol limits biases due to lineage-specific effects on developmental kinetics, proliferation, and apoptosis. Also, lowering the dose of estradiol reduced activated C/EBP␣-ER to near the physiologic range. C/EBP␣-ER increased Mac1 ؉ /Gr1 ؊ /MPO ؊/low monocytes 1.9-fold while reducing Mac1 ؉ /Gr1 ؉ / MPO hi granulocytes 2.5-fold at 48 hours, even in 0.01 M estradiol. This pattern was confirmed morphologically and by quantitative polymerase chain reaction (PCR) assay of lineage markers. To directly assess effects on immature progenitors, transduced cells were cultured for 1 day with and then in methylcellulose without estradiol. A 2-fold increase in monocytic compared with granulocytic colonies was observed in IL-3/IL-6/SCF or GM-CSF, but not G-CSF, even in 0.01 M IntroductionC/EBP␣ and its family members C/EBP␤, C/EBP␦, and C/EBP⑀ are expressed predominantly in myeloid cells within hematopoiesis, with C/EBP␣ the most prominent isoform in immature myeloid cells. 1-3 C/EBPs homodimerize or heterodimerize via C-terminal, ␣-helical leucine zipper (LZ) domains and then contact DNA via the adjacent basic region (BR). 4 C/EBP␣ transactivates lineage-specific genes, inhibits cell cycle progression via interaction with E2F1 and additional mechanisms, and stimulates cell survival by inducing bcl-2 in cooperation with NF-B. [5][6][7] C/EBP␣ Ϫ/Ϫ hematopoietic cells derived from either fetal liver or adult marrow do not effectively generate granulocyte-monocyte progenitors (GMPs) from the common myeloid progenitor (CMP). 8,9 However, the role of C/EBP␣ in the further commitment of the GMP to the granulocytic versus the monocytic myeloid lineages remains uncertain. Exogenous C/EBP␣ directs granulocytic maturation of several myeloid cell lines or erythroid progenitors but induces monocytic development from B-cell progenitors. 3,[10][11][12] Importantly, neither myeloid cell lines nor erythroid or lymphoid progenitors represent the ideal cellular context to investigate the role of C/EBP␣ in myelopoiesis because each may lack critical cytokine signals and cooperating transcription factors. KRAB-C/ EBP␣-estradiol receptor (ER), containing the C/EBP␣ DNAbinding domain linked to the KRAB transrepression domain and the ER ligand-binding domain, markedly suppresses both granulocytic and monocytic marrow colony formation by murine myeloid progenitors. 13 However, this fusion protein may bind and so actively repress genes not physiologically regulated by C/EBP␣.To evaluate the role of C/EBP␣ in monocyte versus granulocyte lineage determination, we have transduced murine myeloid progenitors with C/EBP␣-ER, a fusion protein regulated by estradiol (E 2 ). 14 Marrow cells were subjected to lineage d...

We examined 103 oral squamous cell carcinomas (OSCC), 100 oral lesions consisting primarily of leukoplakia (82 cases), and 76 clinically normal mucosa specimens from the contralateral site in the oral cavity of individuals with oral lesions, for the presence of Epstein-Barr virus (EBV). Polymerase chain reaction (PCR) was used to amplify a 239 bp fragment of the BamHIL region of the EBV genome, followed by Southern blot hybridization with EBV oligonucleotide probe to increase further the specificity and sensitivity of the assay system. Since EBV seropositivity is frequent in populations, we also examined the peripheral blood cells (PBC) from 141 patients (50 oral cancer patients, 91 patients with oral lesions) for the presence of EBV We detected EBV in 25 of 103 (25%) OSCC, 13 of 100 (13%) oral lesions, 3 of 76 (4%) clinically normal mucosa samples and 10 of 141 (7%) PBC. Our results indicate that EBV may contribute as one of the multiple factors in oral cancers, in a certain proportion of Indian patients. z: y ~~~~,~~~t e i n -B a r r virus; Oral cancer;Pare'3 Mumbai 400 012,