In this report, we show that the overexpression of tissue transglutaminase (tTG) in the human neuroblastoma cell line SK-N-BE(2) renders these neural crest-derived cells highly susceptible to death by apoptosis. Cells transfected with a full-length tTG cDNA, under the control of a constitutive promoter, show a drastic reduction in proliferative capacity paralleled by a large increase in cell death rate. The dying tTG-transfected cells exhibit both cytoplasmic and nuclear changes characteristic of cells undergoing apoptosis. The tTG-transfected cells express high Bcl-2 protein levels as well as phenotypic neural cell adhesion molecule markers (NCAM and neurofilaments) of cells differentiating along the neuronal pathway. In keeping with these findings, transfection of neuroblastoma cells with an expression vector containing segments of the human tTG cDNA in antisense orientation resulted in a pronounced decrease of both spontaneous and retinoic acid (RA)-induced apoptosis. We also present evidence that (i) the apoptotic program of these neuroectodermal cells is strictly regulated by RA and (ii) cell death by apoptosis in the human neuroblastoma SK-N-BE(2) cells preferentially occurs in the substrate-adherent phenotype. For the first time, we report here a direct effect of tTG in the phenotypic maturation toward apoptosis. These results indicate that the tTG-dependent irreversible cross-linking of intracellular protein represents an important biochemical event in the induction of the structural changes featuring cells dying by apoptosis.The transglutaminase multigene family includes intracellular and extracellular enzymes that catalyze Ca2"-dependent reac-tions in which -y-carboxamide groups of peptide-bound glutamine residues serve as acyl donors and primary amino groups of several compounds function as acceptor substrates (16,22). The reaction results in posttranslational modification of proteins by establishing e(-y-glutamyl)lysine cross-linkages and/or covalent incorporation of biogenic amines (di-and polyamines and histamine) into proteins. Diamines and polyamines also participate in cross-linking reactions through the formation of N,N-bis(-y-glutamyl)polyamine cross-bridges (16, 40, 43). The transglutaminase-dependent formation of stable cross-linkages induces protein polymerization, which confers high resistance to mechanical breakage and chemical attack to the polypeptides involved in the linkage (17,21

The p53 tumor suppressor gene is one of the most frequently mutated genes in human cancers. 1 p53 is a sequence-specific transcription factor and plays a critical role in activating the expression of genes involved in cell cycle arrest or apoptosis under conditions of genotoxic stress. 2,3 For over two decades, p53 was thought to be the only gene of its kind in the vertebrate genomes. This strong conviction, which was widely accepted in the p53 field, has now been proven to be incorrect. Two genes, referred to as p63 and p73, have been found to encode proteins that share a significant amino-acid identity in the transactivation domain, the DNA binding domain, and the oligomerization domain with p53. In the short period since their cloning, a number of investigators have reported on the structure, the function and the regulation of p63 and p73. This review summarizes the current information on the p63 and the p73 genes, with a focus on the differences between the three members in this newly defined p53-gene family.Keywords: p73; p53; c-Abl; apoptosis Abbreviations: HPV-16, human papilloma virus-16; IGFBP, Insulin-like growth factor binding proteins; IR, ionizing irradiation; MEFs, mouse embryo ®broblasts; MMS, methylmethane sulfonate; OD, oligomerization domain; SAM, sterile alpha motif; TA, transactivation domain; DN p63, N-terminal deleted p63 variants Alternative splicing of p63 and p73The p53 gene generates a single mRNA with a single open reading frame. In contrast, both the p63 and the p73 genes generate several differentially spliced variants. With the p73 gene, alternative splicings not only add or delete coding sequences, but also alter the reading frame. Hence, the p63 and the p73 genes can each encode several different proteins. Most notably, both the p63 and the p73 genes encode alternatively spliced C-terminal regions that are not found in the p53 protein (Figure 1).The p63 gene encodes at least six open reading frames: from the usage of two different promoters/ATG in combination with three alternatively spliced C-terminal ends (Figure 1). The three isoforms (TA-a, TA-b and TAg) are produced by a 5'-promoter and alternative splicing at the 3' end of the gene. These three isoforms contain the coding sequence for the N-terminal transactivation (TA) domain. Each of these three splice variants can also be expressed from an internal promoter upstream of exon 3', that provides a different ATG to initiate translation downstream of the TA domain. These N-terminal deleted p63 isoforms are referred to as DN-alpha, DN-beta and DNgamma. These DN p63 isoforms do not activate transcription but instead can inhibit the transactivation functions of the full length p63 proteins and of p53. 4 The p73 gene generates at least six open reading frames with alternatively spliced 3-region. Initially, two isoforms of p73 were identified: 5 the full length alphaisoform and a C-terminal shortened beta-isoform resulting from the alternative splicing of exon 13. Four other spliced forms of p73 have since been identified in...

Additional complexity in p73: induction by mitogens in lymphoid cells and identification of two new splicing variants e and z Dear Editor, p53 is a sequence specific transcription factor which transactivates several genes important in the apoptotic pathway, such as p21, mdm2, gadd45, bax and caspases. 1,2 Lack of apoptosis, with inappropriate cell proliferation in cancer is correlated with a high frequency of p53 mutations, in some tumours reaching 50%. Perhaps surprisingly, in view of the importance of p53 in regulating cell death and its strong phylogenetic conservation, only recently have homologous genes been identified.These (p63 and p73) show up to 63% aminoacid identity with p53 in the DNA binding, oligomerization and transcription activation domains, suggesting a similar mechanism of action to p53. 3±7 p63 is expressed as six different forms. 4 These use one of the two alternative transcription initiation sites, each transcript then being expressed as one of three alternatively spliced variants (a, b, g). Since the transcripts using the downstream ATG lack the first three exons coding for the transactivation domain, they act as natural dominant negative mutants of full length p63 and of p53. 4 The p73 gene comprises 14 exons and we have shown previously that in addition to the full length a form and the alternatively spliced transcript lacking exon 13 (b) other splice variants, lacking exon 11 (g) and exons 11, 12 and 13 (d) are also produced. 8 Stimulation of the T lymphoblastoid cell line, Jurkat, and human peripheral blood lymphocytes (PBL) with phytoemagglutinin (PHA) causes a 3 and 2.6-fold increase respectively in p73 expression by Northern blotting after 24 h (panel A). This is associated with induction of 34.7% and 21.2% of apoptotic cells respectively. No p53 was detected in Jurkat cells after PHA treatment (not shown).In order to discriminate the differential induction of the four p73 isoforms we performed an RT±PCR using isoformspecific primers on RNA extracted from cells treated under the same conditions. Panel B shows upregulation of a, b, g and d in PHA-treated PBL and Jurkat cells. Densitometric comparison of these with the housekeeping gene GAPDH showed that the increase in expression of each isoform were comparable (not shown).In addition, a new isoform was amplified from normal PBL. Cloning and sequencing of this p73e identified it as a splicing variant lacking exons 11 and 13 (panel C). To confirm the existence of p73e, we screened a panel of normal and tumour cell lines. As also shown in panel B, p73e was also present in the human hepatoma line HepG2, and a sixth isoform z was identified in the MCF7 human breast cancer cell line and in a human skin biopsy. p73 z is a further splice variant which lacks exons 11 and 12, and results in the loss of 96 aminoacids, the sequence continuing with the C-terminus of the a form (panel C). In p73e, loss of exon 11 deletes 50 aminoacids with a frame shift to the reading frame of the g isoform; splicing of exon 13 deletes an additional 31 aminoacids...

Activation of serine biosynthesis supports growth and proliferation of cancer cells. Human cancers often exhibit overexpression of phosphoglycerate dehydrogenase (PHGDH), the metabolic enzyme that catalyses the reaction that diverts serine biosynthesis from the glycolytic pathway. By refueling serine biosynthetic pathways, cancer cells sustain their metabolic requirements, promoting macromolecule synthesis, anaplerotic flux and ATP. Serine biosynthesis intersects glutaminolysis and together with this pathway provides substrates for production of antioxidant GSH. In human lung adenocarcinomas we identified a correlation between serine biosynthetic pathway and p73 expression. Metabolic profiling of human cancer cell line revealed that TAp73 activates serine biosynthesis, resulting in increased intracellular levels of serine and glycine, associated to accumulation of glutamate, tricarboxylic acid (TCA) anaplerotic intermediates and GSH. However, at molecular level p73 does not directly regulate serine metabolic enzymes, but transcriptionally controls a key enzyme of glutaminolysis, glutaminase-2 (GLS-2). p73, through GLS-2, favors conversion of glutamine in glutamate, which in turn drives the serine biosynthetic pathway. Serine and glutamate can be then employed for GSH synthesis, thus the p73-dependent metabolic switch enables potential response against oxidative stress. In knockdown experiment, indeed, TAp73 depletion completely abrogates cancer cell proliferation capacity in serine/glycine-deprivation, supporting the role of p73 to help cancer cells under metabolic stress. These findings implicate p73 in regulation of cancer metabolism and suggest that TAp73 influences glutamine and serine metabolism, affecting GSH synthesis and determining cancer pathogenesis.