Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis.

The antitumor effect and mechanisms activated by murine IL-12 and IL-18, cytokines that induce IFN-gamma production, were studied using engineered SCK murine mammary carcinoma cells. In syngeneic A/J mice, SCK cells expressing mIL-12 or mIL-18 were less tumorigenic and formed tumors more slowly than control cells. Neither SCK.12 nor SCK.18 cells protected significantly against tumorigenesis by distant SCK cells. However, inoculation of the two cell types together synergistically protected 70% of mice from concurrently injected distant SCK cells and 30% of mice from SCK cells established 3 d earlier. Antibody neutralization studies revealed that the antitumor effects of secreted mIL-12 and mIL-18 required IFN-gamma. Interestingly, half the survivors of SCK.12 and/or SCK.18 cells developed protective immunity suggesting that anti-SCK immunity is unlikely to be responsible for protection. Instead, angiogenesis inhibition, assayed by Matrigel implants, appeared to be a property of both SCK.12 and SCK.18 cells and the two cell types together produced significantly greater systemic inhibition of angiogenesis. This suggests that inhibition of tumor angiogenesis is an important part of the systemic antitumor effect produced by mIL-12 and mIL-18.

B7-1 and interleukin 12 synergistically induce effective antitumor immunity.

Enhanced host rejection of tumor cells is the primary goal of cancer immunotherapy and, in many murine tumor models, has been accomplished by engineering cells to express B7 costimulatory molecules or creating an environment rich in certain cytokines. We examined the effect of tumor cell B7-1 expression and administered recombinant interleukin 12 (IL-12) on the syngeneic host response to rapidly growing, poorly immunogenic SCK mammary carcinoma cells and to more slowly growing, immunogenic K1735 melanoma cells. Whereas B7-1 expression induced rejection of K1735 cells in 78% of mice, and IL-12 induced rejection in 38%, B7-1 expression induced rejection of SCK cells in only 28% of mice, and IL-12 induced rejection in none. The relative ineffectiveness of either B7-1 or IL-12 alone to induce rejection of SCK cells led us to combine the two manipulations. This resulted in rejection of SCK cells in 74% of mice and dramatically delayed tumor development in the remainder. Tumor rechallenge studies indicated that the surviving mice developed specific immunity to wild-type SCK cells. Lymphocyte subset ablation and IFN-gamma depletion studies indicated that rejection of SCK tumor cells brought about by the synergistic effects of B7-1 and IL-12 is mediated by a rapidly developing, systemic antitumor immune response that is dependent on the presence of both CD8+ and CD4+ T cells and involves IFN-gamma. Additionally, the synergistic effect of B7-1 expression and IL-12 administration is capable of inducing rejection of control SCK tumors simultaneously established in the opposite flank. The efficacy of B7-1 and IL-12 in inducing protective immunity against a poorly immunogenic, aggressive murine tumor indicates that this combination is particularly effective at producing a potent antitumor immune response that may be of therapeutic benefit.

Recombinant interleukin 12 enhances cellular immune responses to vaccination only after a period of suppression.

Adjuvant use of recombinant murine IL-12 (rmIL-12) was examined in mice vaccinated with irradiated syngeneic tumor or allogeneic cells. rmIL-12 given to A/J mice vaccinated with irradiated SCK tumor cells engineered to secrete granulocyte/macrophage-colony-stimulating factor resulted in significantly better protection from tumor challenges 28 days after vaccination but, unexpectedly, severely compromised host protection 14 days after vaccination. Immune suppression was rmIL-12 dose dependent and manifested as reduced splenic CTL activity, stimulated cytokine release and ability to reject SCK cells. Transient immune suppression was also seen with rmIL-12 given during vaccination of C3H/HeN mice with irradiated K1735 melanoma cells and of C57BL/6 mice with irradiated allogeneic HKB cells. The period of suppression coincided with transiently reduced splenic T-cell mitogenic responses to concanavalin A and IL-2, suggesting that they may be causally related. Suppression appears to be due to impaired immune effector mechanisms rather than impaired host immunization, which is actually enhanced as evidenced by the enhanced reaction to immunogens when hosts are challenged later after rmIL-12 administration. Demonstration that rmIL-12, as it is frequently used, induces a transient period of impaired immune response that can compromise host protection suggests that the unquestioned effectiveness of rmIL-12 against murine tumors is primarily due to activation of mechanisms other than antigen-specific tumor immunity (e.g., antiangiogenic effects) and that use of human IL-12 should be monitored for similar effects.

Critical role of Rho in cell transformation by oncogenic Ras.

We demonstrate that Rho, a regulator of cytoskeletal actin, is necessary for Ras transformation. A dominant inhibitory Rho gene (RhoBN19) specifically suppressed Rat1 cell focus formation induced by oncogenic Ras but not by Raf. An activated Rho gene (RhoBV14) lacked focus formation activity but augmented the focus formation activity of both oncogenes. NIH3T3 cell lines expressing RhoBV14 grew to higher saturation density and displayed reduced serum and anchorage requirements for growth. We concluded that Rho played a role in cell growth regulation and was required for transformation by oncogenic Ras but not Raf. A model for Ras signal transduction proposing separate Rho-dependent and Raf-dependent pathways is discussed.

Effect of the pSV2-neo plasmid on NIH 3T3 cell motion detected electrically.

Advantage was taken of DNA transfection techniques to investigate the effect of the pSV2-neo plasmid and its derivatives on recipient NIH 3T3 cell motion. Cell spreading and motion were followed by a newly developed electrical method to monitor cell morphology, referred to as electric cell-substrate impedance sensing. Using this method, we found that the eukaryotic-prokaryotic shuttle vector pSV2-neo had a strong effect on the recipient NIH 3T3 cell spreading and cell motion. However, two new neo plasmids, pSK-neo and pSP-neo, which were constructed by modifying the pSV2-neo plasmid, did not have a significant effect on the recipient cell activities. The results suggest that there may be some sequences in pSV2-neo which affect recipient cell behavior.