The present studies examined the temporal sequence of inducible nitric oxide synthase (iNOS) gene expression and the cellular sources of iNOS protein and of 3-nitrotyrosine, as a marker of production of nitric oxide-derived reactive nitrogen intermediates during murine multi-stage carcinogenesis. Levels of iNOS mRNA in dorsal skin isolated from acetone-treated female Sencar mice were 2.5-fold higher than iNOS gene expression detected in cutaneous tissue isolated from Sencar mice at 1, 3, 6, 10, 16 and 22 weeks after exposure to a single topical application of 25 nmol 7,12-dimethylbenz[a]anthracene (DMBA) followed by repetitive applications of 2 microgram 17-O-tetradecanoylphorbol-13-acetate (TPA). Papillomas isolated at 16 and 22 weeks of a tumor promotion protocol also had low levels of iNOS mRNA. The diminished levels of iNOS mRNA inversely correlated with the extent of TPA-induced epidermal hyperplasia. In acetone-treated mouse skin, iNOS immunospecific antibody binding was localized to the stratum corneum and suprabasal keratinocytes. In contrast, iNOS protein was present in lower amounts and was localized to the upper-most suprabasal keratinocytes in cutaneous tissue isolated at 22 weeks following a single exposure to either 25 nmol DMBA or acetone and repetitive applications of 2 microgram TPA. At all time points examined over the 22 week time period of papilloma growth, infiltrating neutrophils within the dermis bound significant levels of anti-iNOS antibodies. The production of iNOS by neutrophils within the dermis correlated with the formation of protein nitrotyrosination within the dermal tissue, as detected by 3-nitrotyrosine-specific antibodies. The present studies indicate that NOS and reactive nitrogen intermediates, including peroxynitrite, are produced specifically by dermal neutrophils during the tumor promotion process at time points that correspond to simultaneous production of reactive oxygen intermediates. Conversely, iNOS is simultaneously down-regulated in hyperplastic epidermis and in papillomas.

mRNA for interleukin 7 (IL-7) was readily detected in leukemic cells immediately upon their removal from patients with chronic B-lymphocytic leukemia (B-CLL). IL-7 mRNA expression and IL-7 gene transcription were down regulated, however, when B-CLL cells were placed in culture at 37 degrees C for 4 hr. Down regulation of the IL-7 gene was prevented in cells maintained at 4 degrees C. Continued culture of B-CLL cells at 37 degrees C resulted in programmed cell death, or apoptosis, as evidenced by DNA fragmentation. The coincident kinetics of IL-7 gene down regulation and apoptosis suggested that IL-7 gene expression may be required for maintenance of CLL viability in vivo. Signals for IL-7 gene regulation and apoptosis induction were thus examined. Activation of normal B cells through their immunoglobulin receptors did not result in upregulation of IL-7 gene expression. Reagents required for CLL cell purification and culture also did not contribute to IL-7 gene regulation and apoptosis induction. IL-7 gene expression was retained and apoptosis was prevented, however, in CLL cells cultured on a monolayer of EA.hy926 human umbilical cord endothelial hybrid cells. Signals specifically presented by EA.hy926 cells supported both CLL cell viability and IL-7 gene expression, whereas culture of CLL cells on A549/8 carcinoma cells, the fusion partner used to generate the EA.hy926 cells, did not. Cell-cell contact was required, as culture supernatants did not prevent apoptosis. Specifically, IL-7 mRNA expression was retained and apoptosis was prevented only by contact with the endothelial cell hybrids. Preliminary data indicated that integrins expressed on CLL cells affected modulation of apoptosis and IL-7 gene regulation, suggesting that integrins may play significant roles in regulating viability of CLL cells.

SummaryRegulation of expression of interleukin 7 (IL-7) mRNA is aberrant in the leukemic subset of cells of chronic lymphocytic leukemia (CLL) patients. The entire coding sequence for IL-7 as well as an alternatively spliced IL-7 mRNA are transcribed in these leukemic cells. No IL-7 mRNA expression is detected in fresh peripheral blood mononuclear cells from normal individuals. Furthermore, the "normal" nonleukemic subsets of cells isolated from the same CLL patients also do not express IL-7 mRNA. The only subset of cells in which IL-7 mRNA is detected is the one that contains the leukemic cells themselves. The polymerase chain reaction was used to examine cytokine expression, and flow cytometry was used to purify the various subsets of peripheral blood mononuclear cells examined in these studies, as well as to examine IL-7 receptor expression. A proportion of the cells from the CLL patients express receptors that are capable of binding IL-7, whereas T cell-depleted normal cell preparations do not express receptors for IL-7 that are detectable with IL-7 fluorokines. The IL-7 receptor-bearing cells in CLL patients include a portion of leukemic cells and a fraction of the T cells, as well as some non-T, non-B cells. These findings suggest that IL-7 and IL-7 receptor expression in CLL may be relevant not only to growth regulation of the leukemic cells but to the immunological abnormalities that occur in the disease as well, possibly via the induction of inappropriate immune activity of IL-7 receptor-bearing cells.