A common E2F-1 and p73 pathway mediates cell death induced by TCR activation.

Strong stimulation of the T-cell receptor (TCR) on cycling peripheral T cells causes their apoptosis by a process called TCR-activation-induced cell death (TCR-AICD). TCR-AICD occurs from a late G1 phase cell-cycle check point independently of the 'tumour suppressor' protein p53. Disruption of the gene for the E2F-1 transcription factor, an inducer of apoptosis, causes significant increases in T-cell number and splenomegaly. Here we show that T cells undergoing TCR-AICD induce the p53-related gene p73, another mediator of apoptosis, which is hypermethylated in lymphomas. Introducing a dominant-negative E2F-1 protein or a dominant-negative p73 protein into T cells protects them from TCR-mediated apoptosis, whereas dominant-negative E2F-2, E2F-4 or p53 does not. Furthermore, E2F-1-null or p73-null primary T cells do not undergo TCR-mediated apoptosis either. We conclude that TCR-AICD occurs from a late G1 cell-cycle checkpoint that is dependent on both E2F-1 and p73 activities. These observations indicate that, unlike p53, p73 serves to integrate receptor-mediated apoptotic stimuli.

Transduction of full-length Tat fusion proteins directly into mammalian cells: analysis of T cell receptor activation-induced cell death.

Currently, delivery of expression vectors, proteins, and/or pharmacologically important peptidyl mimetics to target cells is problematic because of the low percentage of cells targeted, overexpression, size constraints, and bioavailability. Concentration-dependent transduction of full-length proteins and domains directly into cells would serve to alleviate these problems. Previous researchers have demonstrated the ability of proteins linked to the human immunodeficiency virus (HIV) Tat transduction domain to transduce into cells; but because of inefficiencies, this methodological potential has not significantly progressed since 1988. We describe, in this chapter, a significant increase in transduction efficiency of proteins and ease of use by (1) generation of a Tat protein transduction domain in-frame bacterial expression vector, pTAT-HA, and (2) development of a purification protocol yielding denatured proteins. We have transduced full-length Tat fusion proteins ranging in size from 15 to 115 kDa into approximately 100% of all target cells examined, including peripheral blood lymphocytes, all cells present in whole blood, bone marrow stem cells, diploid fibroblasts, fibrosarcoma cells, and keratinocytes. Transduction occurs in a concentration-dependent manner, achieving maximum intracellular concentrations in less than 10 min. We conclude that our methodology generates highly efficient transducible proteins that are biologically active and have broad potential in the manipulation of biological experimental systems, such as apoptotic induction, cell cycle progression, and differentiation, and in the delivery of pharmacologically relevant proteins.

Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein.

At present, treatment of HIV infection uses small inhibitory molecules that target HIV protease; however, the emergence of resistant HIV strains is increasingly problematic. To circumvent this, we report here a new 'Trojan horse' strategy to kill HIV-infected cells by exploiting HIV protease. We engineered a transducing, modified, apoptosis-promoting caspase-3 protein, TAT-Casp3, that substitutes HIV proteolytic cleavage sites for endogenous ones and efficiently transduces about 100% of cells, but remains inactive in uninfected cells. In HIV-infected cells, TAT-Casp3 becomes processed into an active form by HIV protease, resulting in apoptosis of the infected cell. This strategy could also be applied to other pathogens encoding specific proteases, such as hepatitis C virus, cytomegalovirus and malaria.

TCR antigen-induced cell death occurs from a late G1 phase cell cycle check point.

Deletion of antigen-activated T cells after an immune response and during peripheral negative selection after strong T cell receptor (TCR) engagement of cycling T cells occurs by an apoptotic process termed TCR antigen-induced cell death (AID). By analyzing the timing of death, cell cycle markers, BrdU-labeled S phase cells, and phase-specific centrifugally elutriated cultures from stimulated Jurkat T cells and peripheral blood lymphocytes, we found that AID occurs from a late G1 check point prior to activation of cyclin E:Cdk2 complexes. T cells stimulated to undergo AID can be rescued by effecting an early G1 block by direct transduction of p16INK4a tumor suppressor protein or by inactivation of the retinoblastoma tumor suppressor protein (pRb) by transduced HPV E7 protein. These results suggest that AID occurs from a late G1 death check point in a pRb-dependent fashion.

Transforming activity and mitosis-related expression of the AKT2 oncogene: evidence suggesting a link between cell cycle regulation and oncogenesis.

The AKT2 oncogene encodes a protein-serine/threonine kinase containing a pleckstrin homology domain characteristic of many signaling proteins. Recently, it was shown that AKT2 kinase activity can be induced by platelet-derived growth factor through phosphatidylinositol-3-OH kinase, suggesting that AKT2 may be an important signal mediator that contributes to the control of cell proliferation. We previously reported amplification and overexpression of AKT2 in human cancers. To investigate the transforming activity of AKT2, we used a retrovirus-based construct to express AKT2 in NIH3T3 cells. Overexpression of AKT2 was found to transform NIH3T3 cells, as determined by growth in soft agar and tumor formation in nude mice. The oncogenic activity of AKT2 was diminished by truncation of a 70-amino acid proline-rich region at the carboxyl-terminus. To facilitate the characterization of AKT2, we generated monoclonal and polyclonal antibodies against this protein. AKT2 was localized to the cytoplasm by cell fractionation experiments, immunocytochemistry, and immunofluorescence. Protein levels were more abundant in mitotic cells than in interphase cells. Western blot analysis of synchronized pancreatic cancer cells demonstrated that the expression level of AKT2 protein in mitotic cells is three to fivefold higher than in their interphase counterparts. A time-course study of phytohemagglutinin-stimulated lymphocytes revealed that AKT2 mRNA and AKT2 protein levels are highest 48-72 h after addition of mitogen, when cells are actively dividing. These data suggest that AKT2 could play a significant role in cell cycle progression and that the oncogenic activity of overexpressed AKT2 may be mediated by aberrant regulation of cellular proliferation.

Isolation, characterization, and mapping to human chromosome 11q24-25 of a cDNA encoding a highly conserved putative transmembrane protein, TMC.

We report the isolation of a novel human cDNA encoding a putative transmembrane protein, TMC. The predicted protein sequence is highly conserved evolutionarily. The cDNA clone was mapped to human chromosome 11q24-25 by fluorescence in situ hybridization. mRNA expression was observed in all tissues tested with the highest levels in testes and ovary.

Identification of zinc finger mRNAs using domain-specific differential display.

An oligonucleotide primer specific for a conserved amino acid region of the Cys2/His2 zinc finger proteins was used in conjunction with mRNA differential display to amplify related mRNAs from a human ovarian cancer cell line. Six of the 12 cDNAs analyzed from the differential display polyacrylamide gel exhibited zinc finger homology at the nucleotide and predicted amino acid sequence level. None of these cDNA fragments, however, shared complete homology with genes encoding any known zinc finger proteins. All 6 cDNA fragments with zinc finger homology had a poly-A tail and 3 of these fragments contained a putative polyadenylation signal. Northern blot analysis was performed using radiolabeled probes prepared from the 12 cDNA fragments. Two of the 6 cDNA fragments with zinc finger homology hybridized to 3.6- and 6.0-kb mRNAs. In addition, 2 of the fragments which did not contain significant homology to zinc finger or any other known sequences hybridized to 4.1- and 5.8-kb mRNAs. These results suggest that domain-specific differential display may be a useful approach for the identification of novel gene family members as well as for the analysis of changes in gene expression of family members between related cell lines or tissue samples.