How do cancer cells acquire the fuel needed to support cell growth?

In this paper we consider whether the dependency of metazoan cells on extracellular signals to maintain cell survival results in an important barrier that must be overcome during carcinogenesis. It is now generally accepted that a major barrier to cancer comes from the inability of cells to enter and progress through the cell cycle in a cell-autonomous fashion. Most of the oncogenes studied over the last two decades contribute to the ability of the cancer cell to enter and progress through the cell cycle in the absence of the instructional signals normally imparted by extracellular growth factors. Over the last two decades, it has begun to be appreciated that there is a second potential barrier to transformation. It appears that all cells in multicellular organisms need extracellular signals not only to initiate proliferation, but also to maintain cell survival. Every cell in our body expresses the proteins necessary to execute its own death by apoptosis. A cell will activate this apoptotic program by default unless it receives signals from the extracellular environment that allow the cell to suppress the apoptotic machinery it expresses. It now appears that the molecular basis of this suppression lies in the signaling pathways that regulate cellular nutrient uptake and direct the metabolic fate of those nutrients.

Deficiency of Bax and Bak protects photoreceptors from light damage in vivo.

Photoreceptors of bax(-/-)bak(-/-) but neither bax(-/-) mice nor bak(-/-) mice are protected from developmental apoptosis, suggesting that bax(-/-)bak(-/-) photoreceptors may also be protected from pathologic apoptosis. To test this possibility, we exposed bax(-/-)bak(-/-) and bax(-/-) mice to bright light, which normally induces photoreceptor death. Photoreceptors in bax(-/-)bak(-/-) mice were protected from death compared to bax(-/-) mice as indicated by a reduction in the number of TUNEL-positive photoreceptor nuclei 24 h following light damage and almost complete preservation of photoreceptors 7 days following light damage. These results provide the first in vivo evidence that combined deficiency of Bax and Bak can rescue cells from a pathologic stimulus more effectively than Bax deficiency and suggest that combined deficiency of Bax and Bak may also protect cells from other insults.

BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis.

Critical issues in apoptosis include the importance of caspases versus organelle dysfunction, dominance of anti- versus proapoptotic BCL-2 members, and whether commitment occurs upstream or downstream of mitochondria. Here, we show cells deficient for the downstream effectors Apaf-1, Caspase-9, or Caspase-3 display only transient protection from "BH3 domain-only" molecules and die a caspase-independent death by mitochondrial dysfunction. Cells with an upstream defect, lacking "multidomain" BAX, BAK demonstrate long-term resistance to all BH3 domain-only members, including BAD, BIM, and NOXA. Comparison of wild-type versus mutant BCL-2, BCL-X(L) indicates these antiapoptotics sequester BH3 domain-only molecules in stable mitochondrial complexes, preventing the activation of BAX, BAK. Thus, in mammals, BH3 domain-only molecules activate multidomain proapoptotic members to trigger a mitochondrial pathway, which both releases cytochrome c to activate caspases and initiates caspase-independent mitochondrial dysfunction.

BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak.

The BH3-only proteins Bim and Bad bind to the antiapoptotic Bcl-2 proteins and induce apoptosis in wild-type cells and cells from either bax(-/-) or bak(-/-) animals. In contrast, constitutively active forms of Bim and Bad failed to induce apoptosis in bax(-/-)bak(-/-) cells. Expression of Bax restored susceptibility of the cells to Bim and Bad. In addition, Bax but not Bim or Bad sensitized the bax(-/-)bak(-/-) cells to a wide variety of cell death stimuli including UV irradiation, chemotherapeutic agents, and ER stress. These results suggest that neither activation of BH3-only proteins nor suppression of pro-survival Bcl-2 proteins is sufficient to kill cells in the absence of both Bax and Bak. Furthermore, whereas mouse embryo fibroblasts (MEF) expressing only Bax or Bak displayed resistance to transformation, bax(-/-)bak(-/-) MEF were nearly as prone to oncogenic transformation as p53(-/-) MEF. Thus, the function of either Bax or Bak appears required to initiate most forms of apoptosis and to suppress oncogenic transformation.

Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.

Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.

Characterization of XIAP-deficient mice.

The inhibitor of apoptosis protein (IAP) family consists of a number of evolutionarily conserved proteins that function to inhibit programmed cell death. X-linked IAP (XIAP) was cloned due to its sequence homology with other family members and has previously been shown to prevent apoptosis by binding to active caspases 3, 7, and 9 in vitro. XIAP transcripts can be found in a variety of tissues, and the protein levels are regulated both transcriptionally and posttranscriptionally. To better understand the function of XIAP in normal cells, we generated mice deficient in XIAP through homologous gene targeting. The resulting mice were viable, and histopathological analysis did not reveal any differences between XIAP-deficient and wild-type mice. We were unable to detect any defects in induction of caspase-dependent or -independent apoptosis in cells from the gene-targeted mice. One change was observed in cells derived from XIAP-deficient mice: the levels of c-IAP1 and c-IAP2 protein were increased. This suggests that there exists a compensatory mechanism that leads to upregulation of other family members when XIAP expression is lost. The changes in c-IAP1 and c-IAP2 expression may provide functional compensation for loss of XIAP during development or in the induction of apoptosis.

BCLW mediates survival of postmitotic Sertoli cells by regulating BAX activity.

Male mice deficient in BCLW, a death-protecting member of the BCL2 family, are sterile due to an arrest in spermatogenesis that is associated with a gradual loss of germ cells and Sertoli cells from the testis. As Bclw is expressed in both Sertoli cells and diploid male germ cells, it has been unclear which of these cell types requires BCLW in a cell-autonomous manner for survival. To determine whether death of Sertoli cells in Bclw mutants is influenced by the protracted loss of germ cells, we examined testes from Bclw/c-kit double mutant mice, which lack germ cells from birth. Loss of BCLW-deficient Sertoli cells occurs in the absence of germ cells, indicating that germ cell death is not required to mediate loss of Sertoli cells in BCLW-deficient mice. This suggests that Sertoli cells require BCLW in a cell-intrinsic manner for long-term survival. The loss of Sertoli cells in Bclw mutants commences shortly after Sertoli cells have become postmitotic. In situ hybridization analysis indicates that Bclw is expressed in Sertoli cells both before and after exit from mitosis. Therefore, Bclw-independent pathways promote the survival of undifferentiated, mitotic Sertoli cells. We show that BAX and BAK, two closely related death-promoting members of the BCL2 family, are expressed in Sertoli cells. To determine whether either BAX or BAK activity is required for Sertoli cell death in Bclw mutant animals, we analyzed survival of Sertoli cells in Bclw/Bax and Bclw/Bak double homozygous mutant mice. While mutation of Bak had no effect, ablation of Bax suppressed the loss of Sertoli cells in Bclw mutants. Thus, BCLW mediates survival of postmitotic Sertoli cells in the mouse by suppressing death-promoting activity of BAX.

tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c.

TNFR1/Fas engagement results in the cleavage of cytosolic BID to truncated tBID, which translocates to mitochondria. Immunodepletion and gene disruption indicate BID is required for cytochrome c release. Surprisingly, the three-dimensional structure of this BH3 domain-only molecule revealed two hydrophobic alpha-helices suggesting tBID itself might be a pore-forming protein. Instead, we demonstrate that tBID functions as a membrane-targeted death ligand in which an intact BH3 domain is required for cytochrome c release, but not for targeting. Bak-deficient mitochondria and blocking antibodies reveal tBID binds to its mitochondrial partner BAK to release cytochrome c, a process independent of permeability transition. Activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux, integrating the pathway from death receptors to cell demise.

TRAF4 deficiency leads to tracheal malformation with resulting alterations in air flow to the lungs.

TRAF4 is one of six identified members of the family of TNFR-associated factors. While the other family members have been found to play important roles in the development and maintenance of a normal immune system, the importance of TRAF4 has remained unclear. To address this issue, we have generated TRAF4-deficient mice. Despite widespread expression of TRAF4 in the developing embryo, as well as in the adult, lack of TRAF4 expression results in a localized, developmental defect of the upper respiratory tract. TRAF4-deficient mice are born with a constricted upper trachea at the site of the tracheal junction with the larynx. This narrowing of the proximal end of the trachea results in respiratory air flow abnormalities and increases rates of pulmonary inflammation. These data demonstrate that TRAF4 is required to regulate the anastomosis of the upper and lower respiratory systems during development.

The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues.

Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. As presented here, bak(-/-) mice were found to be developmentally normal and reproductively fit and failed to develop any age-related disorders. However, when Bak-deficient mice were mated to Bax-deficient mice to create mice lacking both genes, the majority of bax(-/-)bak(-/-) animals died perinatally with fewer than 10% surviving into adulthood. bax(-/-)bak(-/-) mice displayed multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis.

Effects of CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells.

In this report, conditions for prolonged in vitro proliferation of polyclonal adult CD4+ T cells via stimulation with immobilized anti-CD3 plus anti-CD28 have been established. CD4+ cells maintained exponential growth for more than 60 days during which a total 10(9)- to 10(11)-fold expansion occurred. Cell cultures exhibited cyclical changes in cell volume, indicating that, in terms of proliferative rate, cells do not have to rest before restimulation. Indeed, electronic cell size analysis was the most reliable method to determine when to restimulate with additional immobilized mAb. The initial approximately 10(5)-fold expansion was autocrine, occurring in the absence of exogenous cytokines or feeder cells. Addition of recombinant human IL-2 after the initial autocrine expansion resulted in continued exponential proliferation. Phorbol ester plus ionomycin also induced long-term growth when combined with anti-CD28 stimulation. Analysis of the T cell repertoire after prolonged expansion revealed a diverse repertoire as assessed by anti-TCR Vbeta Abs or a PCR-based assay. Cytokines produced were consistent with maintenance of both Th1 and Th2 phenotypes; however, the mode of CD3 and CD28 stimulation could influence the cytokine secretion pattern. When anti-CD3 and anti-CD28 were immobilized on the same surface, ELISAs on culture supernatants revealed a pattern consistent with Th1 secretion. Northern analysis revealed that cytokine gene expression remained inducible. Spontaneous growth or cell transformation was not observed in more than 100 experiments. Together, these observations may have implications for gene therapy and adoptive immunotherapy. Furthermore, these culture conditions establish a model to study the finite lifespan of mature T lymphocytes.

iPABP, an inducible poly(A)-binding protein detected in activated human T cells.

The poly(A)-binding protein (PABP) binds to the poly(A) tail present at the 3' ends of most eukaryotic mRNAs. PABP is thought to play a role in both translation and mRNA stability. Here we describe the molecular cloning and characterization of an inducible PABP, iPABP, from a cDNA library prepared from activated T cells. iPABP shows 79% sequence identity to PABP at the amino acid level. The RNA binding domains of iPABP and PABP are nearly identical, while their C termini are more divergent. Like PABP, iPABP is primarily localized to the cytoplasm. iPABP is expressed at low levels in resting normal human T cells; following T-cell activation, however, iPABP mRNA levels are rapidly up-regulated. In contrast, PABP is constitutively expressed in both resting and activated T cells. iPABP mRNA was also expressed at much higher levels than PABP mRNA in heart and skeletal muscle tissue. These data suggest that the regulation of cytoplasmic poly(A)-binding activity is more complex than previously believed. In most tissues, poly(A)-binding activity is likely to be the result of the combined effects of constitutively expressed PABP and iPABP, whose expression is subject to more complex regulation.

CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL.

T cell activation through the TCR can result in either cell proliferation or cell death. The role of costimulatory receptors in regulating T cell survival has not been defined. Here, we present data demonstrating that CD28 costimulation enhances the in vitro survival of activated T cells. One mechanism for this enhancement is the ability of CD28 costimulation to augment the production of IL-2, which acts as an extrinsic survival factor for T cells. In addition, CD28 costimulation augments the intrinsic ability of T cells to resist apoptosis. Although CD28 signal transduction had no effect on Bcl-2 expression, CD28 costimulation was found to augment the expression of Bcl-XL substantially. Transfection experiments demonstrated that this level of Bcl-XL could prevent T cell death in response to TCR cross-linking, Fas cross-linking, or IL-2 withdrawal. These data suggest that an important role of CD28 costimulation is to augment T cell survival during antigen activation.

Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division.

Growth factors have been defined by their ability to promote the proliferative expansion of receptor-bearing cells. For example, antigen-activated T cells expressing the alpha beta gamma form of the interleukin 2 (IL-2) receptor will proliferate in response to IL-2. In contrast, resting T cells, which express the IL-2 receptor beta and gamma chains, do not proliferate in response to IL-2. We demonstrate that the survival of resting T cells following gamma irradiation is greatly enhanced by pretreatment with IL-2. The radioprotective effect of IL-2 is dose dependent, does not result from the induction of cell proliferation, and does not require expression of the IL-2 receptor alpha chain. Thus, the beta gamma IL-2 receptor expressed on resting T cells can transduce signals that promote cell survival without committing the T cell to undergo cell division. IL-4 and IL-7, but not IL-1, IL-3, or IL-6, were also found to enhance the survival of quiescent T cells following gamma irradiation. Thus, certain growth factor-receptor interactions can serve to maintain cell viability in a manner that is independent of their ability to initiate or maintain cell proliferation. These data may have important implications for the use of growth factors in patients being treated with radiation and/or chemotherapy.

Effects of human T lymphocyte activation on inosine monophosphate dehydrogenase expression.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the first step in the formation of guanine ribonucleotides from inosine monophosphate and the activity of this enzyme appears to be essential for cell proliferation. Inhibitors of IMPDH have been demonstrated to be effective immunosuppressive agents and to inhibit T cell activation in vitro. IMPDH activity results from the expression of two different genes (types I and II) that encode protein subunits of identical size with 84% amino acid identity. To determine the relative contribution of the expression of these two genes to T cell activation, we have examined the effects of T cell stimulation on IMPDH activity, mRNA levels, and protein. The stimulation of isolated peripheral blood CD28+ T cells with PMA and ionomycin causes a 15-fold increase in IMPDH activity over a 72-h period. This is associated with a 10-fold increase in type II mRNA levels at 48 h. Type I mRNA is expressed at very low levels in resting T cells, but increases 10-fold by 24 h after stimulation. The type I cDNA probe also detects a second larger mRNA species of 4.0 kb that is not detectable in a variety of normal tissues or in a panel of leukemic cell lines. RNase protection assays using RNA probes corresponding to the entire coding region of the type I enzyme reveal a single protected fragment, demonstrating that the 4.0-kb message is the result of alternate splicing in the 5' or 3' untranslated regions or the use of an alternative polyadenylation site. Western blot analysis demonstrates a concomitant increase in total IMPDH protein on T cell activation, although posttranslational modifications do not allow the distinction between type I and type II on isoelectric focusing gels. We conclude that the induction of both type I and type II IMPDH contribute significantly to the T cell proliferative response. Both enzymes therefore should be considered important targets for immunosuppressive therapy.

AU-A, an RNA-binding activity distinct from hnRNP A1, is selective for AUUUA repeats and shuttles between the nucleus and the cytoplasm.

The 3'-untranslated regions of many labile transcripts contain AU-rich sequences that serve as cis determinants of mRNA stability and translational efficiency. Using a photocrosslinking technique, our laboratory has previously defined three cytoplasmic RNA-binding activities specific for the AUUUA multimers found in the 3'-untranslated regions of lymphokine mRNAs. One of these activities, AU-A, has an apparent molecular mass of 34 kDa, is constitutively expressed in both primary T cells and the Jurkat T cell leukemia line, and binds to a variety of U-rich RNA sequences. Previous studies had shown that AU-A is more prevalent in the nucleus than the cytoplasm, raising the possibility that AU-A is really a nuclear RNA-binding activity that is found in cytoplasmic extracts because of nuclear leakage during cell fractionation. We now show that AU-A shuttles between the cytoplasm and the nucleus. Our results indicate that AU-A is a candidate protein component of ribonucleoprotein complexes that participate in nucleocytoplasmic transport of mRNA and cytoplasmic mRNA metabolism. The properties of AU-A activity are similar to those of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). However, using monoclonal antibodies to hnRNP A1 and protease digestion patterns, we show that AU-A activity and hnRNP A1 protein are distinct. These studies have also allowed us to define a fourth RNA-binding activity of apparent molecular mass 41 kDa with specificity for AUUUA multimers. This activity is restricted to the nucleus and contains the hnRNP C protein.

Characterization of CTLA-4 structure and expression on human T cells.

CTLA-4 is an adhesion receptor expressed on activated T cells. The amino acid sequence of CTLA-4 is related to CD28, and although the function of CTLA-4 remains unknown, it shares several features with CD28, including a common counter-receptor, B7, that is present on Ag-presenting cells. In a recent study we found that CD28 and CTLA-4 were coexpressed at the mRNA level on activated T cells but that only CD28 was expressed on resting T cells. Here we show that within the T cell population, CTLA-4 expression is restricted to the subset of T cells that also express cell surface CD28. CTLA-4 mRNA expression can be induced on quiescent T cells via phorbol ester-mediated activation of protein kinase C but not with calcium ionophore treatment alone. Phorbol ester-induced expression of CTLA-4 mRNA could be enhanced with calcium ionophore treatment, and treatment of cells in this manner resulted in a reciprocal decrease in expression of CD28 mRNA. Ligation of CD28 with monoclonal antibody also resulted in the specific and rapid induction of CTLA-4 mRNA. To study the expression of CTLA-4 at the protein level, a rabbit antiserum against a recombinant protein derived from CTLA-4 cDNA was generated. When activated T cells were labeled with [35S]methionine, the rabbit antiserum precipitated a 41- to 43-kDa protein from whole cell lysates. Similar results were found when detergent-soluble lysates from 125I surface-labeled resting and activated T cells were analyzed by SDS-PAGE. Surprisingly, under the conditions tested, CTLA-4 migrated primarily as a monomer at the cell surface, and could not be shown to exist as a disulfide-bonded homodimer or as a heterodimer consisting of CTLA-4 and CD28. These results suggest that B7 can bind to T cells via distinct receptor complexes consisting of either CD28 or CTLA-4, and that these complexes may potentially mediate distinct biologic functions. Further, the present results suggest that noncovalent interactions might mediate association of CTLA-4 and/or CD28 at the cell surface.