Lentiviruses infect both dividing and nondividing cells. In in pHP. The ability of this vector system to transduce dividthis study we characterized a lentiviral vector system coning and nondividing cell in vitro and in vivo was also demsisting of a packaging vector (pHP) and a transducing veconstrated. Compared with a Moloney murine leukemia tor (pTV) derived from a recombinant human immunodefivirus (MLV) vector, the HP/TV vectors transduced human ciency virus type 1 (HIV-1). In pHP, the long terminal muscle-, kidney-, liver-derived cell lines and CD34 + primary repeats (LTRs), the 5Ј untranslated leader and portions of hematopoietic progenitor cells more efficiently. Although the env and nef genes were deleted. The leader sequence the levels of the pTV transgene expression were high soon of pHP was substituted with a modified Rous sarcoma virus after transduction, the expression tended to decrease with (RSV) 59 bp leader containing a mutated RSV gag AUG time due either to the loss of proviral DNA or to the inactiand a functional 5Ј splice site. The pHP construct was vation of promoter activity, which was found to be cell typefound to direct Gag-Pol synthesis as efficiently as wild-type dependent. Analyses of extrachromosomal DNA showed HIV-1. The pTV construct contains sequences required for that the unintegrated proviral DNA of lentiviral vectors sur-RNA packaging, reverse transcription and integration, but vived much longer than that of the retroviral vectors. We lacks viral genes. Co-transfection of pHP, pTV and a vesdemonstrate that the HP/TV vector is capable of high icular stomatitis virus G (VSV-G) envelope plasmid proefficiency transduction and that long-term expression of duced vectors at titers of 10 5 -10 6 transducing units per lentiviral vectors is dependent on target cell type, the milliliter in 48 h. Replication-competent virus (RCV) was internal promoter and the transgene itself in the transducnot detected when deletions were made in the env gene ing vector.

Granulocyte-macrophage colony-stimulating activty (GM-CSA) can be produced by a variety of normal cell types including mononuclear phagocytes, activated T lymphocytes, endothelial cells, and fibroblasts. Recent evidence shows that a major role of the monocyte-macrophage is the recruitment of environmental cells, i.e., fibroblasts, to produce GM-CSA. In this study we have identified interleukin 1 (IL-i) as a monokine that stimulates fibroblasts to produce and release GM-CSA and prostaglandin E2 (PGE2). Both purified human monocyte-derived IL-I and human recombinant ILiA (10-10 M) can be substituted for monocyteconditioned medium in stimulating fibroblast GM-CSA and PGE2 production. Both forms of IL-1 stimulate fibroblasts to produ6e GM-CSA and PGE2 in a dose-dependent fashion. The fibroblkst-stimulating activity found in monocyte-conditioned medium was completely blocked by anti-I-iA. We conclude that monocytes produce IL-1, and that monocyte-derived IL-i induces fibroblasts to produce GM-CSA and PGE2.

Aldehyde dehydrogenase (ALDH) activity has been implicated in multiple biological and biochemical pathways and has been used to identify potential cancer stem cells. Our main hypothesis is that ALDH activity may be a lung cancer stem cell marker. Using flow cytometry, we sorted cells with bright (ALDH br ) and dim (ALDH lo ) ALDH activity found in H522 lung cancer cell line. We used in vitro proliferation and colony assays as well as a xenograft animal model to test our hypothesis. Cytogenetic analysis demonstrated that the ALDH br cells are indeed a different clone, but when left in normal culture conditions will give rise to ALDH lo cells. Furthermore, the ALDH br cells grow slower, have low clonal efficiency, and give rise to morphologically distinct colonies. The ability to form primary xenografts in NOD/SCID mice by ALDH br and ALDH lo cells was tested by injecting single cell suspension under the skin in each flank of same animal. Tumor size was calculated weekly. ALDH1A1 and ALDH3A1 immunohistochemistry (IHC) was performed on excised tumors. These tumors were also used to re-establish cell suspension, measure ALDH activity, and re-injection for secondary and tertiary transplants. The results indicate that both cell types can form tumors but the ones from ALDH br cells grew much slower in primary recipient mice. Histologically, there was no significant difference in the expression of ALDH in primary tumors originating from ALDH br or ALDH lo cells. Secondary and tertiary xenografts originating from ALDH br grew faster and bigger than those formed by ALDH lo cells. In conclusion, ALDH br cells may have some of the traditional features of stem cells in terms of being mostly dormant and slow to divide, but require support of other cells (ALDH lo ) to sustain tumor growth. These observations and the known role of ALDH in drug resistance may have significant therapeutic implications in the treatment of lung cancer.

We conclude that both enzymes, ALDH1A1 and ALDH3A1 will need to be blocked in order to achieve the highest sensitivity to 4-HC. Furthermore, ATRA increases 4-HC toxicity even when added to a combination of siRNAs against both enzymes, thus suggesting additional mechanisms by which ATRA can increase drug toxicity.