Our somatic cells are born by mitosis and almost all will die by apoptosis, a physiological process of cellular suicide. Cancers can occur when this balance is disturbed, either by an increase in cell proliferation or a decrease in cell death. The goal of cancer therapy is to promote the death of cancer cells without causing too much damage to normal cells. Our knowledge of the mechanisms of apoptosis has enhanced our understanding of how some cancers originate and progress. It has also revealed that existing cancer therapies can work in two ways, by induction of apoptosis as well as by direct toxicity. In some cases resistance to apoptosis may explain why cancer therapies fail. Novel treatments designed to exploit our knowledge of apoptotic mechanisms are under development to promote apoptosis of cancer cells and limit concurrent death of normal cells.

Apoptosis after growth factor withdrawal or drug treatment is associated with mitochondrial cytochrome c release and activation of Apaf-1 and caspase-9. To determine whether loss of Apaf-1, caspase-2, and caspase-9 prevented death of factor-starved cells, allowing them to proliferate when growth factor was returned, we generated IL-3–dependent myeloid lines from gene-deleted mice. Long after growth factor removal, cells lacking Apaf-1, caspase-9 or both caspase-9 and caspase-2 appeared healthy, retained intact plasma membranes, and did not expose phosphatidylserine. However, release of cytochrome c still occurred, and they failed to form clones when IL-3 was restored. Cells lacking caspase-2 alone had no survival advantage. Therefore, Apaf-1, caspase-2, and caspase-9 are not required for programmed cell death of factor-dependent cells, but merely affect its rate. In contrast, transfection with Bcl-2 provided long-term, clonogenic protection, and could act independently of the apoptosome. Unlike expression of Bcl-2, loss of Apaf-1, caspase-2, or caspase-9 would therefore be unlikely to enhance the survival of cancer cells.

We report the cloning of a novel human activator of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 7 (MKK7). The mRNA for MKK7 is widely expressed in humans and mice and encodes a 47-kDa protein (419 amino acids), as determined by immunoblotting endogenous MKK7 with an antibody raised against its N terminus. The kinase domain of MKK7 is closely related to a Drosophila JNK kinase dHep (69% identity) and to a newly identified ortholog from Caenorhabditis elegans (54% identity), and was more distantly related to MKK4, MKK3, and MKK6. MKK7 phosphorylated and activated JNK1 but failed to activate p38 MAPK in co-expression studies. In hematopoietic cells, endogenous MKK7 was activated by treatment with the growth factor interleukin-3 (but not interleukin-4), or by ligation of CD40, the B-cell antigen receptor, or the receptor for the Fc fragment of immunoglobulin. MKK7 was also activated when cells were exposed to heat, UV irradiation, anisomycin, hyperosmolarity or the pro-inflammatory cytokine tumor necrosis factor-␣. Co-expression of constitutively active mutants of RAS, RAC, or CDC42 in HeLa epithelial cells or of RAC or CDC42 in Ba/F3 factor-dependent hematopoietic cells also activated MKK7, suggesting that MKK7 will be involved in many physiological pathways.

Despite its lack of specificity, the inhibitor SB 203580 has been widely used to implicate p38 mitogen-activated protein kinase (MAPK) in the synthesis of many cytokines. Here we show unequivocally that the production of interleukin (IL)-1␤, IL-6, IL-10, and tumor necrosis factor ␣ (TNF␣) requires p38 MAPK activity by demonstrating that the inhibitory effects of SB 203580 were reversed by expression of an SB 203580-resistant form of p38␣ (SBR-p38␣) that fails to bind to SB 203580. This strategy established the requirement for p38 activity for the lipopolysaccharide-stimulated production of IL-10, IL-1␤, and IL-6 by the monocytic cell WEHI 274 and the production of IL-6 and TNF␣ stimulated by ligation of the Fc-␥ receptor of the mast cell MC/9. Expression of SBR-p38␣ in primary macrophages abrogated the ability of SB 203580 to inhibit the lipopolysaccharide-stimulated production of TNF␣ but not of IL-10. Expression of SBR-p38␣ in primary T lymphocytes abrogated the ability of SB 203580 to inhibit the production of interferon-␥ induced by co-ligation of CD3 and CD28 but not the production of interferon-␥ or IL-10 induced by IL-12. These results suggest that the levels of p38 MAPK activity required for maximal cytokine production vary with different cytokines and stimuli.

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