Neutrophil chemokines secreted by tumor cells mount a lung antimetastatic response during renal cell carcinoma progression.

The mechanism by which renal cell carcinoma (RCC) colonizes the lung microenvironment during metastasis remains largely unknown. To investigate this process, we grafted human RCC cells with varying lung metastatic potential in mice. Gene expression profiling of the mouse lung stromal compartment revealed a signature enriched for neutrophil-specific functions that was induced preferentially by poorly metastatic cells. Analysis of the gene expression signatures of tumor cell lines showed an inverse correlation between metastatic activity and the levels of a number of chemokines, including CXCL5 and IL8. Enforced depletion of CXCL5 and IL8 in these cell lines enabled us to establish a functional link between lung neutrophil infiltration, secretion of chemokines by cancer cells and metastatic activity. We further show that human neutrophils display a higher cytotoxic activity against poorly metastatic cells compared with highly metastatic cells. Together, these results support a model in which neutrophils recruited to the lung by tumor-secreted chemokines build an antimetastatic barrier with loss of neutrophil chemokines in tumor cells acting as a critical rate-limiting step during lung metastatic seeding.

Specific inhibition of protein kinase Cbeta expression by antisense RNA affects the activation of Jurkat T lymphoma cells.

Antisense RNA technology was employed to specifically inhibit the expression of the protein kinase Cbeta (PKCbeta) isoform in Jurkat cells, to explore its influence on the expression of surface antigens (CD69) and the cytokines interleukin-8 (IL-8), tumour necrosis factor (TNF)-alpha and beta, and to characterise its controversial involvement in the expression of IL-2 and its receptor (IL-2R). Transfection of cells with an antisense PKCbeta construct (as-PKCbeta-pREP3) significantly increased IL-2R/CD25 expression in phorbol 12-myristate 13-acetate (PMA)-stimulated as-PKCbeta-pREP3 transfectants, in contrast to Jurkat cells transfected with a control as-PKCalpha-pREP3 plasmid. IL-2 production, in contrast, was strongly inhibited in both transfectant populations stimulated by PMA plus the calcium ionophore ionomycin. Three clones (asb1/asb2/asb3), selected from as-PKCbeta-pREP3 transfectants, showed decreased PKCbeta protein levels (40 percent, 50 percent and 60 percent, respectively, as determined by western blotting) and mRNA levels. The specific inhibition was confirmed in immunoblots for other PKC (alpha, delta, epsilon, gamma, theta, and lambda lambda/tau) isoforms and in immunoprecipitates from representative (c2/asb2) clones. Stimulation of PKCbeta-depleted clones significantly increased CD25 expression but decreased IL-2 production (similarly to as-PKCbeta-pREP3 transfectants) and IL-2 message levels. CD69 expression and IL-8 secretion were significantly decreased, but TNFbeta message levels were highly increased in asb2/asb3 clones, without affecting TNFalpha secretion. Analysis of the mitogen-activated protein kinase (MAP Kinase) signalling pathway showed unaltered extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation but increased activation of c-Jun N-terminal kinase (JNK1) and its substrate, the transcription factor ATF-2 (activated transcription factor-2), which are involved in IL-2 gene expression. Our results revealed new PKCbeta functions, affecting CD69 expression and IL-8 production, and support the requirement for PKCbeta in IL-2 secretion/transcription and IL-2R regulation.

Inhibition of protein kinase C alpha expression by antisense RNA in transfected Jurkat cells.

Taking the antisense approach to inhibit the expression of specific protein kinase C (PKC) isoforms, we investigated the function of PKC alpha in T cell activation by transfecting Jurkat cells with an episomal vector (pREP3) containing a copy of the corresponding gene in the antisense orientation. Transfected Jurkat cells were selected with hygromycin and cloned by limiting dilution. Two (as1/as2) stably transfected antisense PKC alpha-pREP3 clones (as PKC alpha-pREP3) exhibited consistently reductions (76% and 85%, respectively) of PKC alpha levels when analyzed by immunoblotting and immunoprecipitation and also of PKC alpha mRNA (75%, as determined by Northern blotting) when compared to control clones (C1/C2) containing the pREP3 vector alone. The ability of the as-PKC alpha-pREP3 construct to specifically reduce PKC alpha levels in both clones was demonstrated by Western blots probed with antibodies against the PKC beta isozyme (the form structurally more similar to PKC alpha) and other representative isoenzymes expressed in Jurkat cells (PKC delta, epsilon, theta, and mu). Stimulation of transfected Jurkat clones with phorbol-12-myristate-13 alone or in the presence of ionomycin resulted in significant reduction of IL-2R alpha expression, TNF-alpha production, and the induction of transcriptional activity of a pIL-2/Luc construct in both as PKC alpha-reduced clones. The magnitude of these decrements paralleled the reductions of PKC alpha expression. The loss of the effects in clone as1 after a high number of passages correlated with the recovery of normal levels of PKC alpha protein, suggesting a link between these processes. Thus, the findings of this study demonstrate the essential role that PKC alpha plays in major events of the T lymphocyte activation process.