Disruption of RB/E2F-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis.

The retinoblastoma protein (RB) has been proposed to function as a negative regulator of cell proliferation by complexing with cellular proteins such as the transcription factor E2F. To study the biological consequences of the RB/E2F-1 interaction, point mutants of E2F-1 which fail to bind to RB were isolated by using the yeast two-hybrid system. Sequence analysis revealed that within the minimal 18-amino acid peptide of E2F-1 required for RB binding, five residues, Tyr (position 411), Glu (419), and Asp-Leu-Phe (423-425), are critical. These amino acids are conserved among the known E2F family members. While mutation of any of these five amino acids abolished binding to RB, all mutants retained their full transactivation potential. Expression of mutated E2F-1, when compared with that of wild-type, significantly accelerated entry into S phase and subsequent apoptosis. These results provide direct genetic evidence for the biological significance of the RB/E2F interaction and strongly suggest that the interplay between RB and E2F is critical for proper cell cycle progression.

Induction of the WAF1/CIP1 protein and apoptosis in human T-cell leukemia virus type I-transformed lymphocytes after treatment with adriamycin by using a p53-independent pathway.

The WAF1/CIP1 protein has been identified as a downstream mediator of the tumor suppressor p53 in regulating cell cycle progression through a G1-phase check-point. Recent work has implicated the functional status of p53 as a critical determinant in the apoptotic response of certain cell lines to DNA damaging agents. By using human T-cell leukemia virus type I-transformed lymphoid cell lines that differ in their level and function of wild-type p53, we investigated the induction of WAF1/CIP1 and apoptosis after exposure to Adriamycin, a genotoxic agent. We found that regardless of the p53 status in these cell lines, WAF1/CIP1 RNA was rapidly induced in response to Adriamycin treatment. An elevated level of WAF1/CIP1 protein was observed as well. Additionally, we demonstrated that apoptosis was induced in all cell lines analyzed despite some having functionally inactive p53 protein. Our data suggest that a p53-independent pathway may play a role in the apoptotic response observed in some cell lines after exposure to DNA damaging agents.

A Fas-associated protein factor, FAF1, potentiates Fas-mediated apoptosis.

Fas, a member of the tumor necrosis factor receptor family, can induce apoptosis when activated by Fas ligand binding or anti-Fas antibody crosslinking. Genetic studies have shown that a defect in Fas-mediated apoptosis resulted in abnormal development and function of the immune system in mice. A point mutation in the cytoplasmic domain of Fas (a single base change from T to A at base 786), replacing isoleucine with asparagine, abolishes the signal transducing property of Fas. Mice homozygous for this mutant allele (lprcg/lprcg mice) develop lymphadenopathy and a lupus-like autoimmune disease. Little is known about the mechanism of signal transduction in Fas-mediated apoptosis. In this study, we used the two-hybrid screen in yeast to isolate a Fas-associated protein factor, FAF1, which specifically interacts with the cytoplasmic domain of wild-type Fas but not the lprcg-mutated Fas protein. This interaction occurs not only in yeast but also in mammalian cells. When transiently expressed in L cells, FAF1 potentiated Fas-induced apoptosis. A search of available DNA and protein sequence data banks did not reveal significant homology between FAF1 and known proteins. Therefore, FAF1 is an unusual protein that binds to the wild type but not the inactive point mutant of Fas. FAF1 potentiates Fas-induced cell killing and is a candidate signal transducing molecule in the regulation of apoptosis.

A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

Neurotrophin 3 rescues neuronal precursors from apoptosis and promotes neuronal differentiation in the embryonic metanephric kidney.

We analyzed the developmental regulation and role of the neurotrophins during metanephric kidney morphogenesis. RNase protection assay revealed the presence of nerve growth factor, neurotrophin 3 (NT-3), and brain-derived neurotrophic factor mRNAs and the regulation of their expression during embryonic development of rat metanephros. NT-3 induced differentiation (neurite outgrowth) and survival (inhibition of apoptosis) of the neuronal precursors in cultured nephrogenic mesenchymes and neuronal differentiation in cultured whole kidneys, whereas NT-4/5, brain-derived neurotrophic factor, and nerve growth factor were without effect. The neurotrophins did not trigger tubular differentiation of isolated nephrogenic cells, which underwent apoptosis when cultured with or without the neurotrophins. NT-3 is thus an inducer of differentiation and a survival factor for renal neuronal cells, but none of the neurotrophins is a morphogen in kidney tubule induction.

Cloning and characterization of a cellular apoptosis susceptibility gene, the human homologue to the yeast chromosome segregation gene CSE1.

We recently isolated human cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by Pseudomonas exotoxin, Pseudomonas exotoxin-derived immunotoxins, diphtheria toxin, and tumor necrosis factor. We report here that one of these fragments is an antisense fragment of a gene homologous to the essential yeast chromosome segregation gene CSE1. Cloning and analysis of the full-length cDNA of the human CSE1 homologue, which we name CAS for cellular apoptosis susceptibility gene, reveals a protein coding region with similar length (971 amino acids for CAS, 960 amino acids for CSE1) and 59% overall protein homology to the yeast CSE1 protein. The conservation of this gene indicates it has an important function in human cells consistent with the essential role of CSE1 in yeast. CAS is highly expressed in human tumor cell lines and in human testis and fetal liver, tissues that contain actively dividing cells. Furthermore, CAS expression increases when resting human fibroblasts are induced to proliferate and decreases when they are growth-arrested. Thus, CAS appears to play an important role in both toxin and tumor necrosis factor-mediated cell death, as well as in cell proliferation.

Platelet-derived growth factor induces apoptosis in growth-arrested murine fibroblasts.

The platelet-derived growth factor (PDGF) is a potent mitogen for murine fibroblasts. PDGF-stimulated cells express a set of immediate-early-response genes but require additional (progression) factors in serum to progress through the cell cycle. Serum-deprived cells are reversibly arrested in G0 phase and fail to fully traverse the G1 phase of the cell cycle when stimulated by PDGF alone. We now report that serum-deprived normal rat kidney fibroblast (NRK) cells stimulated by either PDGF AA or PDGF BB homodimers undergo apoptotic cell death. Furthermore, we show that epidermal growth factor also induces apoptotic cell death in serum-deprived NRK cells, epidermal growth factor enhances the rate of apoptosis in PDGF-treated cells, and a progression factor (insulin) but not endogenously expressed Bc1-2 fully protects NRK cells from PDGF-stimulated apoptosis. The results indicate that PDGF induces apoptosis in growth-arrested NRK cells and that the inability of NRK cells to transit the G1/S checkpoint is the critical determinant in establishing the genetic program(s) to direct the PDGF signal to apoptosis. The results suggest that polypeptide growth factors in vivo may signal cell fate positively or negatively in settings that limit the potential of cells to completely transit the cell cycle.

Membrane-associated CD19-LYN complex is an endogenous p53-independent and Bc1-2-independent regulator of apoptosis in human B-lineage lymphoma cells.

CD19 receptor is expressed at high levels on human B-lineage lymphoid cells and is physically associated with the Src protooncogene family protein-tyrosine kinase Lyn. Recent studies indicate that the membrane-associated CD19-Lyn receptor-enzyme complex plays a pivotal role for survival and clonogenicity of immature B-cell precursors from acute lymphoblastic leukemia patients, but its significance for mature B-lineage lymphoid cells (e.g., B-lineage lymphoma cells) is unknown. CD19-associated Lyn kinase can be selectively targeted and inhibited with B43-Gen, a CD19 receptor-specific immunoconjugate containing the naturally occurring protein-tyrosine kinase inhibitor genistein (Gen). We now present experimental evidence that targeting the membrane-associated CD19-Lyn complex in vitro with B43-Gen triggers rapid apoptotic cell death in highly radiation-resistant p53-Bax- Ramos-BT B-lineage lymphoma cells expressing high levels of Bcl-2 protein without affecting the Bcl-2 expression level. The therapeutic potential of this membrane-directed apoptosis induction strategy was examined in a scid mouse xenograft model of radiation-resistant high-grade human B-lineage lymphoma. Remarkably, in vivo treatment of scid mice challenged with an invariably fatal number of Ramos-BT cells with B43-Gen at a dose level < 1/10 the maximum tolerated dose resulted in 70% long-term event-free survival. Taken together, these results provide unprecedented evidence that the membrane-associated anti-apoptotic CD19-Lyn complex may be at least as important as Bcl-2/Bax ratio for survival of lymphoma cells.

Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis.

The transcription factor GATA-1 recognizes a consensus motif present in regulatory regions of numerous erythroid-expressed genes. Mouse embryonic stem cells lacking GATA-1 cannot form mature red blood cells in vivo. In vitro differentiation of GATA-1- embryonic stem cells gives rise to a population of committed erythroid precursors that exhibit developmental arrest and death. We show here that the demise of GATA-1- erythroid cells is accompanied by several features characteristics of apoptosis. This process occurs despite normal expression of all known GATA target genes examined, including the erythropoietin receptor, and independent of detectable accumulation of the tumor suppressor protein p53. Thus, in addition to its established role in regulating genes that define the erythroid phenotype, GATA-1 also supports the viability of red cell precursors by suppressing apoptosis. These results illustrate the multifunctional nature of GATA-1 and suggest a mechanism by which other hematopoietic transcription factors may ensure the development of specific lineages.

A mutant p53 antagonizes the deregulated c-myc-mediated enhancement of apoptosis and decrease in leukemogenicity.

Myeloid leukemic M1 cells that do not express p53 and transfected M1 clones that constitutively express the [Val135]p53 mutant or deregulated c-myc or coexpressing both genes grew autonomously in culture with a similar growth rate and cloning efficiency. Expression of deregulated c-myc in M1 leukemic cells enhanced susceptibility to induction of apoptotic cell death and resulted in a reduced leukemogenicity when injected into isologous mice. Expression of the [Val135]p53 mutant did not change cell susceptibility to induction of apoptosis or leukemogenicity, but expression of this mutant p53 suppressed the effects of deregulated c-myc on these properties. The results indicate that the [Val135]p53 mutant can show a gain of function for susceptibility to apoptosis and leukemogenicity in leukemic cells with deregulated c-myc and, thus, enhance tumor development.

Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis.

DNA-damaging agents induce accumulation of the tumor suppressor and G1 checkpoint protein p53, leading cells to either growth arrest in G1 or apoptosis (programmed cell death). The p53-dependent G1 arrest involves induction of p21 (also called WAF1/CIP1/SDI1), which prevents cyclin kinase-mediated phosphorylation of retinoblastoma protein (RB). Recent studies suggest a p53-independent G1 checkpoint as well; however, little is known about its molecular mechanisms. We report that induction of a protein-serine/threonine phosphatase activity by DNA damage signals is at least one of the mechanisms responsible for p53-independent, RB-mediated G1 arrest and consequent apoptosis. When two p53-null human leukemic cell lines (HL-60 and U-937) were treated with a variety of anticancer agents, RB became hypophosphorylated, accompanied with G1 arrest. This was followed immediately (in less than 30 min) by apoptosis, as determined by the accumulation of pre-G1 apoptotic cells and the internucleosomal fragmentation of DNA. Addition of calyculin A or okadaic acid (specific serine/threonine phosphatase inhibitors) or zinc chloride (apoptosis inhibitor) prevented the G1 arrest- and apoptosis-specific RB dephosphorylation. The levels of cyclin E- and cyclin A-associated kinase activities remained high during RB dephosphorylation, supporting the involvement of a chemotherapy-induced serine/threonine phosphatase(s) rather than p21. Furthermore, the induced phosphatase activity coimmunoprecipitated with the hyperphosphorylated RB and was active in a cell-free system that reproduced the growth arrest- and apoptosis-specific RB dephosphorylation, which was inhibitable by calyculin A but not zinc. We propose that the RB phosphatase(s) might be one of the p53-independent G1 checkpoint regulators.

Tumor necrosis factor-induced apoptosis is mediated by a CrmA-sensitive cell death pathway.

We report here that the activation of the interleukin 1 beta (IL-1 beta)-converting enzyme (ICE) family is likely to be one of the crucial events of tumor necrosis factor (TNF) cytotoxicity. The cowpox virus CrmA protein, a member of the serpin superfamily, inhibits the enzymatic activity of ICE and ICE-mediated apoptosis. HeLa cells overexpressing crmA are resistant to apoptosis induced by Ice but not by Ich-1, another member of the Ice/ced-3 family of genes. We found that the CrmA-expressing HeLa cells are resistant to TNF-alpha/cycloheximide (CHX)-induced apoptosis. Induction of apoptosis in HeLa cells by TNF-alpha/CHX is associated with secretion of mature IL-1 beta, suggesting that an IL-1 beta-processing enzyme, most likely ICE itself, is activated by TNF-alpha/CHX stimulation. These results suggest that one or more members of the ICE family sensitive to CrmA inhibition are activated and play a critical role in apoptosis induced by TNF.

The human papilloma virus 16E6 gene sensitizes human mammary epithelial cells to apoptosis induced by DNA damage.

Programmed cell death (apoptosis) is a normal physiological process, which could in principle be manipulated to play an important role in cancer therapy. The key importance of p53 expression in the apoptotic response to DNA-damaging agents has been stressed because mutant or deleted p53 is so common in most kinds of cancer. An important strategy, therefore, is to find ways to induce apoptosis in the absence of wild-type p53. In this paper, we compare apoptosis in normal human mammary epithelial cells, in cells immortalized with human papilloma virus (HPV), and in mammary carcinoma cell lines expressing wild-type p53, mutant p53, or no p53 protein. Apoptosis was induced with mitomycin C (MMC), a DNA cross-linking and damaging agent, or with staurosporine (SSP), a protein kinase inhibitor. The normal and HPV-transfected cells responded more strongly to SSP than did the tumor cells. After exposure to MMC, cells expressing wild-type p53 underwent extensive apoptosis, whereas cells carrying mutated p53 responded weakly. Primary breast cancer cell lines null for p53 protein were resistant to MMC. In contrast, two HPV immortalized cell lines in which p53 protein was destroyed by E6-modulated ubiquitinylation were highly sensitive to apoptosis induced by MMC. Neither p53 mRNA nor protein was induced in the HPV immortalized cells after MMC treatment, although p53 protein was elevated by MMC in cells with wild-type p53. Importantly, MMC induced p21 mRNA but not p21 protein expression in the HPV immortalized cells. Thus, HPV 16E6 can sensitize mammary epithelial cells to MMC-induced apoptosis via a p53- and p21-independent pathway. We propose that the HPV 16E6 protein modulates ubiquitin-mediated degradation not only of p53 but also of p21 and perhaps other proteins involved in apoptosis.

Nonsteroidal antiinflammatory drugs cause apoptosis and induce cyclooxygenases in chicken embryo fibroblasts.

Programmed cell death (apoptosis) is an intrinsic part of organismal development and aging. Here we report that many nonsteroidal antiinflammatory drugs (NSAIDs) cause apoptosis when applied to v-src-transformed chicken embryo fibroblasts (CEFs). Cell death was characterized by morphological changes, the induction of tissue transglutaminase, and autodigestion of DNA. Dexamethasone, a repressor of cyclooxygenase (COX) 2, neither induced apoptosis nor altered the NSAID effect. Prostaglandin E2, the primary eicosanoid made by CEFs, also failed to inhibit apoptosis. Expression of the protooncogene bcl-2 is very low in CEFs and is not altered by NSAID treatment. In contrast, p20, a protein that may protect against apoptosis when fibroblasts enter G0 phase, was strongly repressed. The NSAID concentrations used here transiently inhibit COXs. Nevertheless, COX-1 and COX-2 mRNAs and COX-2 protein were induced. In some cell types, then, chronic NSAID treatment may lead to increased, rather than decreased, COX activity and, thus, exacerbate prostaglandin-mediated inflammatory effects. The COX-2 transcript is a partially spliced and nonfunctional form previously described. Thus, these findings suggest that COXs and their products play key roles in preventing apoptosis in CEFs and perhaps other cell types.

Apoptosis induced by a human milk protein.

To the breast-fed infant, human milk is more than a source of nutrients; it furnishes a wide array of molecules that restrict microbes, such as antibodies, bactericidins, and inhibitors of bacterial adherence. However, it has rarely been considered that human milk may also contain substances bioactive toward host cells. While investigating the effect of human milk on bacterial adherence to a human lung cancer cell line, we were surprised to discover that the milk killed the cells. Analysis of this effect revealed that a component of milk in a particular physical state--multimeric alpha-lact-albumin--is a potent Ca(2+)-elevating and apoptosis-inducing agent with broad, yet selective, cytotoxic activity. Multimeric alpha-lactalbumin killed all transformed, embryonic, and lymphoid cells tested but spared mature epithelial elements. These findings raise the possibility that milk contributes to mucosal immunity not only by furnishing antimicrobial molecules but also by policing the function of lymphocytes and epithelium. Finally, analysis of the mechanism by which multimeric alpha-lactalbumin induces apoptosis in transformed epithelial cells could lead to the design of antitumor agents.