Highly Stoichiometric, Stable, and Specific Association of Integrin α3β1 with CD151 Provides a Major Link to Phosphatidylinositol 4-Kinase, and May Regulate Cell Migration

Here we describe an association between α3β1 integrin and transmembrane-4 superfamily (TM4SF) protein CD151. This association is maintained in relatively stringent detergents and thus is remarkably stable in comparison with previously reported integrin–TM4SF protein associations. Also, the association is highly specific (i.e., observed in vitro in absence of any other cell surface proteins), and highly stoichiometric (nearly 90% of α3β1 associated with CD151). In addition, α3β1 and CD151 appeared in parallel on many cell lines and showed nearly identical skin staining patterns. Compared with other integrins, α3β1 exhibited a considerably higher level of associated phosphatidylinositol-4-kinase (PtdIns 4-kinase) activity, most of which was removed upon immunodepletion of CD151. Specificity for CD151 and PtdIns 4-kinase association resided in the extracellular domain of α3β1, thus establishing a novel paradigm for the specific recruitment of an intracellular signaling molecule. Finally, antibodies to either CD151 or α3β1 caused a ∼88–92% reduction in neutrophil motility in response to f-Met-Leu-Phe on fibronectin, suggesting an functionally important role of these complexes in cell migration.

Cell-Adhesive Responses to Tenascin-C Splice Variants Involve Formation of Fascin Microspikes

Tenascin-C is an adhesion-modulating matrix glycoprotein that has multiple effects on cell behavior. Tenascin-C transcripts are expressed in motile cells and at sites of tissue modeling during development, and alternative splicing generates variants that encode different numbers of fibronectin type III repeats. We have examined the in vivo expression and cell adhesive properties of two full-length recombinant tenascin-C proteins: TN-190, which contains the eight constant fibronectin type III repeats, and TN-ADC, which contains the additional AD2, AD1, and C repeats. In situ hybridization with probes specific for the AD2, AD1, and C repeats shows that these splice variants are expressed at sites of active tissue modeling and fibronectin expression in the developing avian feather bud and sternum. Transcripts incorporating the AD2, AD1, and C repeats are present in embryonic day 10 wing bud but not in embryonic day 10 lung. By using a panel of nine cell lines in attachment assays, we have found that C2C12, G8, and S27 myoblastic cells undergo concentration-dependent adhesion to both variants, organize actin microspikes that contain the actin-bundling protein fascin, and do not assemble focal contacts. On a molar basis, TN-ADC is more active than TN-190 in promoting cell attachment and irregular cell spreading. The addition of either TN-190 or TN-ADC in solution to C2C12, COS-7, or MG-63 cells adherent on fibronectin decreases cell attachment and results in decreased organization of actin microfilament bundles, with formation of cortical membrane ruffles and retention of residual points of substratum contact that contain filamentous actin and fascin. These data establish a biochemical similarity in the processes of cell adhesion to tenascin-C and thrombospondin-1, also an “antiadhesive” matrix component, and also demonstrate that both the adhesive and adhesion-modulating properties of tenascin-C involve similar biochemical events in the cortical cytoskeleton. In addition to these generic properties, TN-ADC is less active in adhesion modulation than TN-190. The coordinated expression of different tenascin-C transcripts during development may, therefore, provide appropriate microenvironments for regulated changes in cell shape, adhesion, and movement.

T Cell Receptor-induced Activation and Apoptosis In Cycling Human T Cells Occur throughout the Cell Cycle

Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0–G1, S, and G2–M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca2+ flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle.

Glutathione synthesis is essential for mouse development but not for cell growth in culture

Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of γ-glutamylcysteine synthetase (γGCS-HStm1), an essential enzyme in GSH synthesis. Embryos homozygous for γGCS-HStm1 fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

AIS is an oncogene amplified in squamous cell carcinoma

We and others recently isolated a human p53 homologue (p40/p51/p63/p73L) and localized the gene to the distal long arm of chromosome 3. Here we sought to examine the role of p40/p73L, two variants lacking the N-terminal transactivation domain, in cancer. Fluorescent in situ hybridization (FISH) analysis revealed frequent amplification of this gene locus in primary squamous cell carcinoma of the lung and head and neck cancer cell lines. (We named this locus AIS for amplified in squamous cell carcinoma.) Furthermore, amplification of the AIS locus was accompanied by RNA and protein overexpression of a variant p68AIS lacking the terminal transactivation domain. Protein overexpression in primary lung tumors was limited to squamous cell carcinoma and tumors known to harbor a high frequency of p53 mutations. Overexpression of p40AIS in Rat 1a cells led to an increase in soft agar growth and tumor size in mice. Our results support the idea that AIS plays an oncogenic role in human cancer.

The Gfi-1 protooncoprotein represses Bax expression and inhibits T-cell death

The Gfi-1 protooncogene encodes a nuclear zinc-finger protein that carries a novel repressor domain, SNAG, and functions as a position- and orientation-independent active transcriptional repressor. The Gfi-1 repressor allows interleukin 2 (IL-2)-dependent T cells to escape G1 arrest induced by IL-2 withdrawal in culture and collaborates with c-myc and pim-1 for the induction of retrovirus-induced lymphomas in animals. Here we show that overexpression of Gfi-1 also inhibits cell death induced by cultivation of IL-2-dependent T-cell lines in IL-2-deficient media. Similarly, induction of Gfi-1 in primary thymocytes from mice carrying a metal-inducible Gfi-1 transgene inhibits cell death induced by cultivation in vitro. The protein and mRNA levels of the proapoptotic regulator Bax are down-regulated by Gfi-1 in both immortalized T-cell lines and primary transgenic thymocytes. The repression is direct and depends on several Gfi-1-binding sites in the p53-inducible Bax promoter. In addition to Bax, Gfi-1 also represses Bak, another apoptosis-promoting member of the Bcl-2 gene family. Therefore, Gfi-1 may inhibit apoptosis by means of its repression of multiple proapoptotic regulators. The antiapoptotic properties of Gfi-1 provide a potential explanation for its strong collaboration with c-myc during oncogenesis.

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.

Fas-dependent CD4+ cytotoxic T-cell-mediated pathogenesis during virus infection

β2-Microglobulin-deficient (β2m−) mice generate a CD4+ major histocompatibility complex class II-restricted cytotoxic T-lymphocyte (CTL) response following infection with lymphocytic choriomeningitis (LCM) virus (LCMV). We have determined the cytotoxic mechanism used by these CD4+ CTLs and have examined the role of this cytotoxic activity in pathogenesis of LCM disease in β2m− mice. Lysis of LCMV-infected target cells by CTLs from β2m− mice is inhibited by addition of soluble Fas-Ig fusion proteins or by pretreatment of the CTLs with the protein synthesis inhibitor emetine. In addition, LCMV-infected cell lines that are resistant to anti-Fas-induced apoptosis are refractory to lysis by these virus-specific CD4+ CTLs. These data indicate that LCMV-specific CD4+ CTLs from β2m− mice use a Fas-dependent lytic mechanism. Intracranial (i.c.) infection of β2m− mice with LCMV results in loss of body weight. Fas-deficient β2m−.lpr mice develop a similar wasting disease following i.c. infection. This suggests that Fas-dependent cytotoxicity is not required for LCMV-induced weight loss. A potential mediator of this chronic wasting disease is tumor necrosis factor (TNF)-α, which is produced by LCMV-specific CD4+ CTLs. In contrast to LCMV-induced weight loss, lethal LCM disease in β2m− mice is dependent on Fas-mediated cytotoxicity. Transfer of immune splenocytes from LCMV-infected β2m− mice into irradiated infected β2m− mice results in death of recipient animals. In contrast, transfer of these splenocytes into irradiated infected β2m−.lpr mice does not cause death. Thus a role for CD4+ T-cell-mediated cytotoxicity in virus-induced immunopathology has now been demonstrated.

DNA damage and p53-mediated cell cycle arrest: A reevaluation

Most mammalian cells exhibit transient delays in the G1 and G2 phases of the cell cycle after treatment with radiation or radiomimetic compounds. p53 is required for the arrest in G1, which provides time for DNA repair. Recently, a role of p53 in the G2/M transition has also been suggested. However, it has been reported that the presence of functional p53 does not always correlate with the induction of these checkpoints. To precisely assess the role of p53 in activating cell cycle checkpoints and in cell survival after radiation, we studied the response of two isogenic human fibrosarcoma cell lines differing in their p53 status (wild type or mutant). We found that when irradiated cells undergo a wild-type p53-dependent G1 arrest, they do not subsequently arrest in G2. Moreover, wild-type p53 cells irradiated past the G1 checkpoint arrest in G2 but do not delay in the subsequent G1 phase. Furthermore, in these cell lines, which do not undergo radiation-induced apoptosis, the wild-type p53 cell line exhibited a greater radioresistance in terms of clonogenic survival. These results suggest that the two checkpoints may be interrelated, perhaps through a control system that determines, depending on the extent of the damage, whether the cell needs to arrest cell cycle progression at the subsequent checkpoint for further repair. p53 could be a crucial component of this control system.

Characterization of avian T-cell receptor γ genes

In birds and mammals T cells develop along two discrete pathways characterized by expression of either the αβ or the γδ T-cell antigen receptors (TCRs). To gain further insight into the evolutionary significance of the γδ T-cell lineage, the present studies sought to define the chicken TCRγ locus. A splenic cDNA library was screened with two polymerase chain reaction products obtained from genomic DNA using primers for highly conserved regions of TCR and immunoglobulin genes. This strategy yielded cDNA clones with characteristics of mammalian TCR γ chains, including canonical residues considered important for proper folding and stability. Northern blot analysis with the TCRγ cDNA probe revealed 1.9-kb transcripts in the thymus, spleen, and a γδ T-cell line, but not in B or αβ T-cell lines. Three multimember Vγ subfamilies, three Jγ gene segments, and a single constant region Cγ gene were identified in the avian TCRγ locus. Members of each of the three Vγ subfamilies were found to undergo rearrangement in parallel during the first wave of thymocyte development. TCRγ repertoire diversification was initiated on embryonic day 10 by an apparently random pattern of V-Jγ recombination, nuclease activity, and P- and N-nucleotide additions to generate a diverse repertoire of avian TCRγ genes early in ontogeny.

PTGF-β, a type β transforming growth factor (TGF-β) superfamily member, is a p53 target gene that inhibits tumor cell growth via TGF-β signaling pathway

Identification and characterization of p53 target genes would lead to a better understanding of p53 functions and p53-mediated signaling pathways. Two putative p53 binding sites were identified in the promoter of a gene encoding PTGF-β, a type β transforming growth factor (TGF-β) superfamily member. Gel shift assay showed that p53 bound to both sites. Luciferase-coupled transactivation assay revealed that the gene promoter was activated in a p53 dose- as well as p53 binding site-dependent manner by wild-type p53 but not by several p53 mutants. The p53 binding and transactivation of the PTGF-β promoter was enhanced by etoposide, a p53 activator, and was largely blocked by a dominant negative p53 mutant. Furthermore, expression of endogenous PTGF-β was remarkably induced by etoposide in p53-positive, but not in p53-negative, cell lines. Finally, the conditioned medium collected from PTGF-β-overexpressing cells, but not from the control cells, suppressed tumor cell growth. Growth suppression was not, however, seen in cells that lack functional TGF-β receptors or Smad4, suggesting that PTGF-β acts through the TGF-β signaling pathway. Thus, PTGF-β, a secretory protein, is a p53 target that could mediate p53-induced growth suppression in autocrinal as well as paracrinal fashions. The finding made a vertical connection between p53 and TGF-β signaling pathways in controlling cell growth and implied a potential important role of p53 in inflammation regulation via PTGF-β.

The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions

Lentiviruses, including HIV-1, have transmembrane envelope (Env) glycoproteins with cytoplasmic tails that are quite long compared with those of other retroviruses. However, mainly because of the lack of biochemical studies performed in cell types that are targets for HIV-1 infection, no clear consensus exists regarding the function of the long lentiviral Env cytoplasmic tail in virus replication. In this report, we characterize the biological and biochemical properties of an HIV-1 mutant lacking the gp41 cytoplasmic tail. We find that the gp41 cytoplasmic tail is necessary for the efficient establishment of a productive, spreading infection in the majority of T cell lines tested, peripheral blood mononuclear cells, and monocyte-derived macrophages. Biochemical studies using a high-level, transient HIV-1 expression system based on pseudotyping with the vesicular stomatitis virus glycoprotein demonstrate that in HeLa and MT-4 cells, mutant Env incorporation into virions is reduced only 3-fold relative to wild type. In contrast, gp120 levels in virions produced from a number of other T cell lines and primary macrophages are reduced more than 10-fold by the gp41 truncation. The Env incorporation defect imposed by the cytoplasmic tail truncation is not the result of increased shedding of gp120 from virions or reduced cell-surface Env expression. These results demonstrate that in the majority of T cell lines, and in primary cell types that serve as natural targets for HIV-1 infection in vivo, the gp41 cytoplasmic tail is essential for efficient Env incorporation into virions.

Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control

Numerous studies have implicated the pRB family of nuclear proteins in the control of cell cycle progression. Although over-expression experiments have revealed that each of these proteins, pRB, p107, and p130, can induce a G1 cell cycle arrest, mouse knockouts demonstrated distinct developmental requirements for these proteins, as well as partial functional redundancy between family members. To study the mechanism by which the closely related pRB family proteins contribute to cell cycle progression, we generated 3T3 fibroblasts derived from embryos that lack one or more of these proteins (pRB−/−, p107−/−, p130−/−, pRB−/−/p107−/−, pRB−/−/p130−/−, and p107−/−/p130−/−). By comparing the growth and cell cycle characteristics of these cells, we have observed clear differences in the manner in which they transit through the G1 and S phases as well as exit from the cell cycle. Deletion of Rb, or more than one of the family members, results in a shortening of G1 and a lengthening of S phase, as well as a reduction in growth factor requirements. In addition, the individual cell lines showed differential regulation of a subset of E2F-dependent gene promoters, as well as differences in cell cycle-dependent kinase activity. Taken together, these observations suggest that the closely related pRB family proteins affect cell cycle progression through distinct biochemical mechanisms and that their coordinated action may contribute to their diverse functions in various physiological settings.

Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation

Histone deacetylases (HDACs) catalyze the removal of acetyl groups on the amino-terminal lysine residues of core nucleosomal histones. This activity is associated generally with transcriptional repression. We have reported previously that inhibition of HDAC activity by hydroxamic acid-based hybrid polar compounds, such as suberoylanilide hydroxamic acid (SAHA), induces differentiation and/or apoptosis of transformed cells in vitro and inhibits tumor growth in vivo. SAHA is a potentially new therapeutic approach to cancer treatment and is in Phase I clinical trials. In several tumor cell lines examined, HDAC inhibitors alter the expression of less than 1% of expressed genes, including the cell cycle kinase inhibitor p21WAF1. In T24 bladder carcinoma cells, SAHA induces up to a 9-fold increase in p21WAF1 mRNA and protein, which is, at least in part, because of an increase in the rate of transcription of the gene. SAHA causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin by 2 h, which is maintained through 24 h of culture. An increase in the accumulation of acetylated H3 and H4 was detected throughout the p21WAF1 promoter and the structural gene after culture with SAHA. The level of histone acetylation did not change in chromatin associated with the actin and p27 genes, and their mRNA expression was not altered during culture of T24 cells with SAHA. Thus, the present findings indicate that the induction of p21WAF1 by SAHA is regulated, at least in part, by the degree of acetylation of the gene-associated histones and that this induced increase in acetylation is gene selective.

Human interleukin-10-related T cell-derived inducible factor: Molecular cloning and functional characterization as an hepatocyte-stimulating factor

IL-10-related T cell-derived inducible factor (IL-TIF or IL-21) is a new cytokine structurally related to IL-10 and originally identified in the mouse as a gene induced by IL-9 in T cells and mast cells. Here, we report the cloning of the human IL-TIF cDNA, which shares 79% amino acid identity with mouse IL-TIF and 25% identity with human IL-10. Recombinant human IL-TIF was found to activate signal transducer and activator of transcription factors-1 and -3 in several hepatoma cell lines. IL-TIF stimulation of HepG2 human hepatoma cells up-regulated the production of acute phase reactants such as serum amyloid A, α1-antichymotrypsin, and haptoglobin. Although IL-10 and IL-TIF have distinct activities, antibodies directed against the β chain of the IL-10 receptor blocked the induction of acute phase reactants by IL-TIF, indicating that this chain is a common component of the IL-10 and IL-TIF receptors. Similar acute phase reactant induction was observed in mouse liver upon IL-TIF injection, and IL-TIF expression was found to be rapidly increased after lipopolysaccharide (LPS) injection, suggesting that this cytokine contributes to the inflammatory response in vivo.